CAZypedia - User contributions [en-ca]

Carbohydrate Binding Module Family 73

2018-09-25T11:18:26Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>. The CBM73 module of the tri-modular LPMO, ''Cj''LPMO10A, binds tightly to α- chitin (Kd = 4.3 µM) and binding equilibrium is established within 15 minutes after mixing the protein and substrate <cite>Forsberg2016</cite>.

== Structural Features ==
Currently no NMR or crystal structure is available for CBM73s, but circular dichroism experiments of the CBM from the ''Vibrio cholera'' GlcNAc-binding protein (GbpA, VcLPMO10B) indicated a β-sheet containing structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to the [[CBM5]] family ([[Carbohydrate-binding_modules#Types|type A]]). Despite low sequence similarity, conserved aromatic amino acids within the [[CBM5]] family, responsible for substrate-binding <cite>Akagi2006</cite>, align well with similar residues in the CBM73 family (Figure 1A)<cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in the CBM73 family compared to the CBM5 family. Compatibly, a somewhat lower ''K''d for α-chitin was observed for the C-terminal CBM73 of the ''Cellvibrio japonicus'' LPMO (''Cj''LPMO10A) relative to its internal CBM5 (Figure 1BC)<cite>Forsberg2016</cite>.

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from ''Cellvibrio japonicus'' (Cj), ''Cellvibrio mixus'' (Cm) and ''Streptomyces griseus'' (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of ''C. japonicus'' CBM73 containing enzymes. (C) Comparative binding of the CBMs from the ''C. japonicus'' LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite> , determined by solid state depletion assays. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
Family 73 CBMs are found in Gram-negative bacteria from the phylum of Proteobacteria and are covalently attached to chitin degrading enzymes such as [[GH18]] and [[GH19]] chitinases <cite>Wong2012 Deboy2008</cite>, [[AA10]] chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a [[CBM5]] chitin-binding module. In chitin degrading enzymes from ''C. japonicus'', the family 73 CBMs are found internally as well as at the N- or C-terminus (Figure 1B). The CBM73 from ''Cj''LPMO10A, together with the [[CBM5]], strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs ([[CBM5]] and CBM73) in ''Cj''LPMO10A reduces the lifetime of the catalytic [[AA10]] domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from ''Vibrio vulnificus''. Truncation of the two CBMs ([[CBM5]] and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular ''Cellvibrio japonicus'' chitin-oxidizing LPMO ([[AA10]]-[[CBM5]]-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-09-25T11:16:59Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>. The CBM73 module of the tri-modular LPMO ,''Cj''LPMO10A, binds tightly to α- chitin (Kd = 4.3 µM) and binding equilibrium is established within 15 minutes after mixing the protein and substrate <cite>Forsberg2016</cite>.

== Structural Features ==
Currently no NMR or crystal structure is available for CBM73s, but circular dichroism experiments of the CBM from the ''Vibrio cholera'' GlcNAc-binding protein (GbpA, VcLPMO10B) indicated a β-sheet containing structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to the [[CBM5]] family ([[Carbohydrate-binding_modules#Types|type A]]). Despite low sequence similarity, conserved aromatic amino acids within the [[CBM5]] family, responsible for substrate-binding <cite>Akagi2006</cite>, align well with similar residues in the CBM73 family (Figure 1A)<cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in the CBM73 family compared to the CBM5 family. Compatibly, a somewhat lower ''K''d for α-chitin was observed for the C-terminal CBM73 of the ''Cellvibrio japonicus'' LPMO (''Cj''LPMO10A) relative to its internal CBM5 (Figure 1BC)<cite>Forsberg2016</cite>.

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from ''Cellvibrio japonicus'' (Cj), ''Cellvibrio mixus'' (Cm) and ''Streptomyces griseus'' (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of ''C. japonicus'' CBM73 containing enzymes. (C) Comparative binding of the CBMs from the ''C. japonicus'' LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite> , determined by solid state depletion assays. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
Family 73 CBMs are found in Gram-negative bacteria from the phylum of Proteobacteria and are covalently attached to chitin degrading enzymes such as [[GH18]] and [[GH19]] chitinases <cite>Wong2012 Deboy2008</cite>, [[AA10]] chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a [[CBM5]] chitin-binding module. In chitin degrading enzymes from ''C. japonicus'', the family 73 CBMs are found internally as well as at the N- or C-terminus (Figure 1B). The CBM73 from ''Cj''LPMO10A, together with the [[CBM5]], strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs ([[CBM5]] and CBM73) in ''Cj''LPMO10A reduces the lifetime of the catalytic [[AA10]] domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from ''Vibrio vulnificus''. Truncation of the two CBMs ([[CBM5]] and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular ''Cellvibrio japonicus'' chitin-oxidizing LPMO ([[AA10]]-[[CBM5]]-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-09-25T11:10:17Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>. The CBM73 module of the tri-modular LPMO ''Cj''LPMO10A binds tightly to α- chitin (Kd = 4.3 µM) and binding equilibrium is established within 15 minutes after mixing the protein and substrate <cite>Forsberg2016</cite>.

== Structural Features ==
Currently no NMR or crystal structure is available for CBM73s, but circular dichroism experiments of the CBM from the ''Vibrio cholera'' GlcNAc-binding protein (GbpA, VcLPMO10B) indicated a β-sheet containing structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to the [[CBM5]] family ([[Carbohydrate-binding_modules#Types|type A]]). Despite low sequence similarity, conserved aromatic amino acids within the [[CBM5]] family, responsible for substrate-binding <cite>Akagi2006</cite>, align well with similar residues in the CBM73 family (Figure 1A)<cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in the CBM73 family compared to the CBM5 family. Compatibly, a somewhat lower ''K''d for α-chitin was observed for the C-terminal CBM73 of the ''Cellvibrio japonicus'' LPMO (''Cj''LPMO10A) relative to its internal CBM5 (Figure 1BC)<cite>Forsberg2016</cite>.

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from ''Cellvibrio japonicus'' (Cj), ''Cellvibrio mixus'' (Cm) and ''Streptomyces griseus'' (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of ''C. japonicus'' CBM73 containing enzymes. (C) Comparative binding of the CBMs from the ''C. japonicus'' LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite> , determined by solid state depletion assays. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
Family 73 CBMs are found in Gram-negative bacteria from the phylum of Proteobacteria and are covalently attached to chitin degrading enzymes such as [[GH18]] and [[GH19]] chitinases <cite>Wong2012 Deboy2008</cite>, [[AA10]] chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a [[CBM5]] chitin-binding module. In chitin degrading enzymes from ''C. japonicus'', the family 73 CBMs are found internally as well as at the N- or C-terminus (Figure 1B). The CBM73 from ''Cj''LPMO10A, together with the [[CBM5]], strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs ([[CBM5]] and CBM73) in ''Cj''LPMO10A reduces the lifetime of the catalytic [[AA10]] domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from ''Vibrio vulnificus''. Truncation of the two CBMs ([[CBM5]] and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular ''Cellvibrio japonicus'' chitin-oxidizing LPMO ([[AA10]]-[[CBM5]]-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-09-25T11:05:47Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.Using a solid state depletion assay, the CBM73 module of the tri-modular LPMO ''Cj''LPMO10A was shown to have a dissociation constant (Kd) of 4.3 µM for binding to α- chitin <cite>Forsberg2016</cite>.

== Structural Features ==
Currently no NMR or crystal structure is available for CBM73s, but circular dichroism experiments of the CBM from the ''Vibrio cholera'' GlcNAc-binding protein (GbpA, VcLPMO10B) indicated a β-sheet containing structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to the [[CBM5]] family ([[Carbohydrate-binding_modules#Types|type A]]). Despite low sequence similarity, conserved aromatic amino acids within the [[CBM5]] family, responsible for substrate-binding <cite>Akagi2006</cite>, align well with similar residues in the CBM73 family (Figure 1A)<cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in the CBM73 family compared to the CBM5 family. Compatibly, a somewhat lower ''K''d for α-chitin was observed for the C-terminal CBM73 of the ''Cellvibrio japonicus'' LPMO (''Cj''LPMO10A) relative to its internal CBM5 (Figure 1BC)<cite>Forsberg2016</cite>.

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from ''Cellvibrio japonicus'' (Cj), ''Cellvibrio mixus'' (Cm) and ''Streptomyces griseus'' (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of ''C. japonicus'' CBM73 containing enzymes. (C) Comparative binding of the CBMs from the ''C. japonicus'' LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite> , determined by solid state depletion assays. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
Family 73 CBMs are found in Gram-negative bacteria from the phylum of Proteobacteria and are covalently attached to chitin degrading enzymes such as [[GH18]] and [[GH19]] chitinases <cite>Wong2012 Deboy2008</cite>, [[AA10]] chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a [[CBM5]] chitin-binding module. In chitin degrading enzymes from ''C. japonicus'', the family 73 CBMs are found internally as well as at the N- or C-terminus (Figure 1B). The CBM73 from ''Cj''LPMO10A, together with the [[CBM5]], strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs ([[CBM5]] and CBM73) in ''Cj''LPMO10A reduces the lifetime of the catalytic [[AA10]] domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from ''Vibrio vulnificus''. Truncation of the two CBMs ([[CBM5]] and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular ''Cellvibrio japonicus'' chitin-oxidizing LPMO ([[AA10]]-[[CBM5]]-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-09-25T07:43:28Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no NMR or crystal structure is available for CBM73s, but circular dichroism experiments of the CBM from the ''Vibrio cholera'' GlcNAc-binding protein (GbpA, VcLPMO10B) indicated a β-sheet containing structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to the [[CBM5]] family ([[Carbohydrate-binding_modules#Types|type A]]). Despite low sequence similarity, conserved aromatic amino acids within the [[CBM5]] family, responsible for substrate-binding <cite>Akagi2006</cite>, align well with similar residues in the CBM73 family (Figure 1A)<cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in the CBM73 family compared to the CBM5 family. Compatibly, a somewhat lower ''K''d for α-chitin was observed for the C-terminal CBM73 of the ''Cellvibrio japonicus'' LPMO (''Cj''LPMO10A) relative to its internal CBM5 (Figure 1BC)<cite>Forsberg2016</cite>.

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from ''Cellvibrio japonicus'' (Cj), ''Cellvibrio mixus'' (Cm) and ''Streptomyces griseus'' (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of ''C. japonicus'' CBM73 containing enzymes. (C) Comparative binding of the CBMs from the ''C. japonicus'' LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite> , determined by solid state depletion assays. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
Family 73 CBMs are found in Gram-negative bacteria from the phylum of Proteobacteria and are covalently attached to chitin degrading enzymes such as [[GH18]] and [[GH19]] chitinases <cite>Wong2012 Deboy2008</cite>, [[AA10]] chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a [[CBM5]] chitin-binding module. In chitin degrading enzymes from ''C. japonicus'', the family 73 CBMs are found internally as well as at the N- or C-terminus (Figure 1B). The CBM73 from ''Cj''LPMO10A, together with the [[CBM5]], strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs ([[CBM5]] and CBM73) in ''Cj''LPMO10A reduces the lifetime of the catalytic [[AA10]] domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from ''Vibrio vulnificus''. Truncation of the two CBMs ([[CBM5]] and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular ''Cellvibrio japonicus'' chitin-oxidizing LPMO ([[AA10]]-[[CBM5]]-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-09-18T13:30:49Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no NMR or crystal structure is available for CBM73s, but circular dichroism experiments of the CBM from the ''Vibrio cholera'' GlcNAc-binding protein (GbpA, VcLPMO10B) indicated a β-sheet containing structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (type A). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (''Cj''LPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from ''Cellvibrio japonicus'' (Cj), ''Cellvibrio mixus'' (Cm) and ''Streptomyces griseus'' (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of ''C. japonicus'' CBM73 containing enzymes. (C) Comparative binding of the CBMs from the ''C. japonicus'' LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite>. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the phylum of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2008</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from ''C. japonicus'', the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from ''Cj''LPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in ''Cj''LPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from ''Vibrio vulnificus''. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular ''Cellvibrio japonicus'' chitin-oxidizing LPMO (AA10-CBM5-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-09-18T13:28:56Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no NMR or crystal structure is available for CBM73s, but circular dichroism experiments of the CBM from the ''Vibrio cholera'' GlcNAc-binding protein (GbpA, VcLPMO10B) indicated a β-sheet containing structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (type A). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (''Cj''LPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from ''Cellvibrio japonicus'' (Cj), ''Cellvibrio mixus'' (Cm) and ''Streptomyces griseus'' (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of ''C. japonicus'' CBM73 containing enzymes. (C) Comparative binding of the CBMs from the ''C. japonicus'' LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite>. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2008</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from ''C. japonicus'', the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from ''Cj''LPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in ''Cj''LPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from ''Vibrio vulnificus''. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular ''Cellvibrio japonicus'' chitin-oxidizing LPMO (AA10-CBM5-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-09-18T13:25:34Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no NMR or crystal structure is available for CBM73s, but circular dichroism experiments of the CBM from the ''Vibrio cholera'' GlcNAc-binding protein (GbpA, VcLPMO10B) indicated a β-sheet containing structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (''Cj''LPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from ''Cellvibrio japonicus'' (Cj), ''Cellvibrio mixus'' (Cm) and ''Streptomyces griseus'' (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of ''C. japonicus'' CBM73 containing enzymes. (C) Comparative binding of the CBMs from the ''C. japonicus'' LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite>. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2008</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from ''C. japonicus'', the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from ''Cj''LPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in ''Cj''LPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from ''Vibrio vulnificus''. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular ''Cellvibrio japonicus'' chitin-oxidizing LPMO (AA10-CBM5-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-08-31T09:56:17Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no NMR or crystal structure is available for CBM73s, but circular dichroism experiments of the CBM from the ''Vibrio cholera'' GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (''Cj''LPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from ''Cellvibrio japonicus'' (Cj), ''Cellvibrio mixus'' (Cm) and ''Streptomyces griseus'' (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of ''C. japonicus'' CBM73 containing enzymes. (C) Comparative binding of the CBMs from the ''C. japonicus'' LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite>. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2008</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from ''C. japonicus'', the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from ''Cj''LPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in ''Cj''LPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from ''Vibrio vulnificus''. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular ''Cellvibrio japonicus'' chitin-oxidizing LPMO (AA10-CBM5-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-04-17T12:21:22Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no NMR or crystal structure is available for CBM73s, but circular dichroism experiments of the CBM from the ''Vibrio cholera'' GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (''Cj''LPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from ''Cellvibrio japonicus'' (Cj), ''Cellvibrio mixus'' (Cm) and ''Streptomyces griseus'' (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of ''C. japonicus'' CBM73 containing enzymes. (C) Comparative binding of the CBMs from the ''C. japonicus'' LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite>. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2006</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from ''C. japonicus'', the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from ''Cj''LPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in ''Cj''LPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from ''Vibrio vulnificus''. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular ''Cellvibrio japonicus'' chitin-oxidizing LPMO (AA10-CBM5-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-04-17T12:18:52Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no NMR or crystal structure is available for CBM73s, but circular dichroism experiments of the CBM from the Vibrio cholera GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (CjLPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from Cellvibrio japonicus (Cj), Cellvibrio mixus (Cm) and Streptomyces griseus (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of C. japonicus CBM73 containing enzymes. (C) Comparative binding of the CBMs from the C. japonicus LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite>. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2006</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from C. japonicus, the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from CjLPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in CjLPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from Vibrio vulnificus. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular Cellvibrio japonicus chitin-oxidizing LPMO (AA10-CBM5-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-04-17T12:18:02Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no crystal structure or NMR data is available for CBM73s, but circular dichroism experiments of the CBM from the Vibrio cholera GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (CjLPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from Cellvibrio japonicus (Cj), Cellvibrio mixus (Cm) and Streptomyces griseus (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of C. japonicus CBM73 containing enzymes. (C) Comparative binding of the CBMs from the C. japonicus LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite>. The figure was adapted from Forsberg et al. <cite>Forsberg2016</cite>. ]]

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2006</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from C. japonicus, the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from CjLPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in CjLPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from Vibrio vulnificus. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular Cellvibrio japonicus chitin-oxidizing LPMO (AA10-CBM5-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-04-17T12:14:13Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no crystal structure or NMR data is available for CBM73s, but circular dichroism experiments of the CBM from the Vibrio cholera GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (CjLPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

[[Image:CBM73 fig1.png|thumb|right|600px|'''Figure 1.''' (A) Multiple sequence alignment of CBM5s (grey) and CBM73s (yellow) from Cellvibrio japonicus (Cj), Cellvibrio mixus (Cm) and Streptomyces griseus (Sg). Conserved aromatic residues are labelled white on a blue background and the disulfide in CBM5s are shown above the alignment (S-S). (B) Modular organization of C. japonicus CBM73 containing enzymes. (C) Comparative binding of the CBMs from the C. japonicus LPMO (CjLPMO10A). Dissociation constants of 5.3 µM and 4.3 µM was obtained for α-chitin for the CBM5 and the CBM73, respectively <cite>Forsberg2016</cite>. The figure was adapted from Forsberg et al., <cite>Forsberg2016</cite>. ]]

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2006</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from C. japonicus, the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from CjLPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in CjLPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from Vibrio vulnificus. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular Cellvibrio japonicus chitin-oxidizing LPMO (AA10-CBM5-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

File:CBM73 fig1.png

2018-04-17T12:12:05Z

Gustav Vaaje-Kolstad:

Carbohydrate Binding Module Family 73

2018-04-17T12:04:32Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no crystal structure or NMR data is available for CBM73s, but circular dichroism experiments of the CBM from the Vibrio cholera GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (CjLPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2006</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from C. japonicus, the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from CjLPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in CjLPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from Vibrio vulnificus. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Family CBM73 was first found as a C-terminal module of the tri-modular Cellvibrio japonicus chitin-oxidizing LPMO (AA10-CBM5-CBM73) <cite>Forsberg2016</cite>.

;First Structural Characterization
:Hitherto, no structural information is available

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-04-17T12:03:34Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no crystal structure or NMR data is available for CBM73s, but circular dichroism experiments of the CBM from the Vibrio cholera GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (CjLPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2006</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from C. japonicus, the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from CjLPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in CjLPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from Vibrio vulnificus. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Insert archetype here, possibly including ''very brief'' synopsis.
;First Structural Characterization
:Insert archetype here, possibly including ''very brief'' synopsis.

== References ==
<biblio>
#Forsberg2016 pmid=26858252
#Wong2012 pmid=22253590
#Tuveng2016 pmid=27169553
#Akagi2006 pmid=16567413
#Deboy2008 pmid=18556790
#Lim2011 pmid=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-04-17T12:02:25Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no crystal structure or NMR data is available for CBM73s, but circular dichroism experiments of the CBM from the Vibrio cholera GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (CjLPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2006</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from C. japonicus, the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from CjLPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in CjLPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from Vibrio vulnificus. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Insert archetype here, possibly including ''very brief'' synopsis.
;First Structural Characterization
:Insert archetype here, possibly including ''very brief'' synopsis.

== References ==
<biblio>
#Forsberg2016 PMID=26858252
#Wong2012 PMID=22253590
#Tuveng2016 PMID=27169553
#Akagi2006 PMID=16567413
#Deboy2008 PMID=18556790
#Lim2011 PMID=21642466
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-04-17T12:01:43Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no crystal structure or NMR data is available for CBM73s, but circular dichroism experiments of the CBM from the Vibrio cholera GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (CjLPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2006</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from C. japonicus, the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from CjLPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in CjLPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from Vibrio vulnificus. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Insert archetype here, possibly including ''very brief'' synopsis.
;First Structural Characterization
:Insert archetype here, possibly including ''very brief'' synopsis.

== References ==
<biblio>
#Forsberg2016 PMID=26858252
#Wong2012 PMID=22253590
#Tuveng2016 PMID=27169553
#Akagi2006 PMID=16567413
#Deboy2008 PMID=18556790
#Lim2011 PMID=21642466

</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-04-17T12:00:57Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==
Currently no crystal structure or NMR data is available for CBM73s, but circular dichroism experiments of the CBM from the Vibrio cholera GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (CjLPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite> (Fig. 1).

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2006</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from C. japonicus, the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from CjLPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in CjLPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from Vibrio vulnificus. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.

== Family Firsts ==
;First Identified
:Insert archetype here, possibly including ''very brief'' synopsis.
;First Structural Characterization
:Insert archetype here, possibly including ''very brief'' synopsis.

== References ==
<biblio>
#Cantarel2009 pmid=18838391
#DaviesSinnott2008 Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. ''The Biochemist'', vol. 30, no. 4., pp. 26-32. [http://www.biochemist.org/bio/03004/0026/030040026.pdf Download PDF version].
#Boraston2004 pmid=15214846
#Hashimoto2006 pmid=17131061
#Shoseyov2006 pmid=16760304
#Guillen2010 pmid=19908036
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-04-17T11:59:36Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==

Currently no crystal structure or NMR data is available for CBM73s, but circular dichroism experiments of the CBM from the Vibrio cholera GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (CjLPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite (Fig. 1).

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2006</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from C. japonicus, the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from CjLPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in CjLPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from Vibrio vulnificus. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.
== Family Firsts ==
;First Identified
:Insert archetype here, possibly including ''very brief'' synopsis.
;First Structural Characterization
:Insert archetype here, possibly including ''very brief'' synopsis.

== References ==
<biblio>
#Cantarel2009 pmid=18838391
#DaviesSinnott2008 Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. ''The Biochemist'', vol. 30, no. 4., pp. 26-32. [http://www.biochemist.org/bio/03004/0026/030040026.pdf Download PDF version].
#Boraston2004 pmid=15214846
#Hashimoto2006 pmid=17131061
#Shoseyov2006 pmid=16760304
#Guillen2010 pmid=19908036
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Carbohydrate Binding Module Family 73

2018-04-17T11:58:18Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Zarah Forsberg^^^
* [[Responsible Curator]]: ^^^Gustav Vaaje-Kolstad^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}CBM73.html
|}
</div>


== Ligand specificities ==
Family 73 CBMs are modules of approximately 60 residues that are appended to bacterial enzymes associated to chitin degradation <cite>Forsberg2016 Wong2012 Tuveng2016</cite>. Binding to amorphous and crystalline α- and β-chitin has been demonstrated <cite>Forsberg2016 Wong2012</cite>.

== Structural Features ==Currently no crystal structure or NMR data is available for CBM73s, but circular dichroism experiments of the CBM from the Vibrio cholera GlcNAc-binding protein (GbpA, VcLPMO10B) revealed a β secondary structure <cite>Wong2012</cite>. Sequence alignment shows that the CBM73 are distantly related to chitin-binding modules belonging to Family 5 CBMs (that are Type A CBMs). Despite low sequence similarity, conserved aromatic amino acids of the CBM5s responsible for substrate-binding <cite>Akagi2006</cite> align well with similar residues in the CBM73s <cite>Forsberg2016 Tuveng2016</cite>. Two additional aromatic residues are found in CBM73s compared to CBM5s. Compatibly, a somewhat lower Kd for α-chitin was observed for the C-terminal CBM73 of the Cellvibrio japonicus LPMO (CjLPMO10A) relative to its internal CBM5 <cite>Forsberg2016</cite (Fig. 1).

== Functionalities ==
CBM73s are found in Gram-negative bacteria from the genus of Proteobacteria and are covalently attached to chitin degrading enzymes such as GH18 and GH19 chitinases <cite>Wong2012 Deboy2006</cite>, AA10 chitin-oxidizing lytic polysaccharide monooxygenases <cite>Forsberg2016 Wong2012</cite> and often in combination with a CBM5 chitin-binding module. In chitin degrading enzymes from C. japonicus, the CBM73s are found internally as well as in the N- or C-terminus (Fig. 1). The CBM73 from CjLPMO10A (together with the CBM5) strongly promotes targeting and binding of crystalline α- and β-chitin as the LPMO domain alone binds weakly to its substrate. Removal of the two CBMs (CBM5 and CBM73) in CjLPMO10A reduces the lifetime of the catalytic AA10 domain and decreases the overall product yield. A CBM73 has also been found appended to a serine protease/peptidoglycan hydrolase from Vibrio vulnificus. Truncation of the two CBMs (CBM5 and CBM73) resulted in reduced peptidoglycan hydrolyzing activity but did not affect the protease activity <cite>Lim2011</cite>.
== Family Firsts ==
;First Identified
:Insert archetype here, possibly including ''very brief'' synopsis.
;First Structural Characterization
:Insert archetype here, possibly including ''very brief'' synopsis.

== References ==
<biblio>
#Cantarel2009 pmid=18838391
#DaviesSinnott2008 Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. ''The Biochemist'', vol. 30, no. 4., pp. 26-32. [http://www.biochemist.org/bio/03004/0026/030040026.pdf Download PDF version].
#Boraston2004 pmid=15214846
#Hashimoto2006 pmid=17131061
#Shoseyov2006 pmid=16760304
#Guillen2010 pmid=19908036
</biblio>

[[Category:Carbohydrate Binding Module Families|CBM073]]

Auxiliary Activity Family 10

2018-01-19T07:14:03Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc-containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically active AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 binds chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_scheme_LPMOs.png|thumb|right|600px|'''Figure 2. Reaction schemes proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead to spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functionality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard conditions (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite>). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 M-1 s-1.

== Catalytic Residues and copper coordination ==

[[Image:His_brace_4ALC_label.png|thumb|left|400px|''''''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are shown as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low radiation dose X-ray crystallographic experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial", although this is not always formally correct (as exemplified by Fig. 3). One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

[[Image:AA10_diversityPNG.PNG|thumb|right|600px|'''''''''Figure 4. LPMO active sites.''' Active sites and their "second shell" of residues surrounding the histidine brace. CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite> ]]
The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure 4 shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> describes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO. The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by the Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely involved in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with a copper ion bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as discussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

Auxiliary Activity Family 10

2018-01-19T07:10:29Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc-containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically active AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 binds chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_scheme_LPMOs.png|thumb|right|600px|'''Figure 2. Reaction schemes proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead to spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functionality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard conditions (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite>). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 M-1 s-1.

== Catalytic Residues and copper coordination ==

[[Image:His_brace_4ALC_label.png|thumb|left|400px|''''''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are shown as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low X-ray radiation experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial", although this is not always formally correct (as exemplified by Fig. 3). One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

[[Image:AA10_diversityPNG.PNG|thumb|right|600px|'''''''''Figure 4. LPMO active sites.''' Active sites and their "second shell" of residues surrounding the histidine brace. CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite> ]]
The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure 4 shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> describes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO. The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by the Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely involved in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with a copper ion bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as discussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

Auxiliary Activity Family 10

2018-01-19T06:53:41Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg 2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc-containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically active AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 binds chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_scheme_LPMOs.png|thumb|right|600px|'''Figure 2. Reaction schemes proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead to spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functionality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard conditions (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite>). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 M-1 s-1.

== Catalytic Residues and copper coordination ==

[[Image:His_brace_4ALC_label.png|thumb|left|400px|''''''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are shown as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low X-ray radiation experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial", although this is not always formally correct (as exemplified by Fig. 3). One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

[[Image:AA10_diversityPNG.PNG|thumb|right|600px|'''''''''Figure 4. LPMO active sites.''' Active sites and their "second shell" of residues surrounding the histidine brace. CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite> ]]
The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure 4 shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> describes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO. The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by the Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely involved in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with a copper ion bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as discussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

Auxiliary Activity Family 10

2018-01-19T06:52:32Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg 2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc-containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically active AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 binds chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_scheme_LPMOs.png|thumb|right|600px|'''Figure 2. Reaction schemes proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead to spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functionality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard conditions (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite>). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 M-1 s-1.

== Catalytic Residues and copper coordination ==

[[Image:His_brace_4ALC_label.png|thumb|left|400px|''''''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are shown as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low X-ray radiation experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial", although this is not always formally correct (as exemplified by Fig. 3). One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

[[Image:AA10_diversityPNG.PNG|thumb|right|600px|'''''''''Figure 4. LPMO active sites.''' Active sites and their "second shell" of CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite> ]]
The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure 4 shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> describes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO. The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by the Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely involved in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with a copper ion bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as discussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

File:Rx scheme LPMOs.png

2018-01-19T06:52:19Z

Gustav Vaaje-Kolstad: Reaction scheme of LPMOs from 2010 and 2017

Reaction scheme of LPMOs from 2010 and 2017

Auxiliary Activity Family 10

2018-01-16T13:55:00Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg 2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically actove AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 bind chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_mech.png|thumb|right|600px|'''Figure 2. Reaction mechanisms proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2 - showing the old and the new equation). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functionality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard conditions (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite> MORE REFs needed?). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 m-1 s-1.

== Catalytic Residues and copper coordination ==

[[Image:His_brace_4ALC_label.png|thumb|left|400px|''''''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are showed as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low X-ray radiation experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial". One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

[[Image:AA10_diversityPNG.PNG|thumb|right|600px|'''''''''Figure 4. LPMO active sites.''' Active sites and their "second shell" of CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite> ]]
The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure 4 shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> descibes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely invovled in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with the copper ion (that is essential for activity) bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as dicussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

Auxiliary Activity Family 10

2018-01-16T13:52:42Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg 2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically actove AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 bind chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_mech.png|thumb|right|400px|'''Figure 2. Reaction mechanisms proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2 - showing the old and the new equation). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functionality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard conditions (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite> MORE REFs needed?). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 m-1 s-1.

== Catalytic Residues and copper coordination ==

[[Image:His_brace_4ALC_label.png|thumb|left|400px|''''''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are showed as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low X-ray radiation experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial". One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure 4 shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> descibes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

[[Image:AA10_diversityPNG.PNG|thumb|right|400px|'''''''''Figure 4. LPMO active sites.''' Active sites and their "second shell" of CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite> ]]
== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely invovled in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with the copper ion (that is essential for activity) bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as dicussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

Auxiliary Activity Family 10

2018-01-16T13:50:27Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg 2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically actove AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 bind chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_mech.png|thumb|right|400px|'''Figure 2. Reaction mechanisms proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2 - showing the old and the new equation). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functionality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard conditions (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite> MORE REFs needed?). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 m-1 s-1.

== Catalytic Residues and copper coordination ==

[[Image:His_brace_4ALC_label.png|thumb|left|400px|''''''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are showed as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low X-ray radiation experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial". One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure 4 shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> descibes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

[[Image:AA10_diversityPNG.png|thumb|right|400px|'''''''''Figure 4. LPMO active sites.''' Active sites and their "second shell" of CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite> ]]
== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely invovled in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with the copper ion (that is essential for activity) bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as dicussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

Auxiliary Activity Family 10

2018-01-16T13:49:42Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg 2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically actove AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 bind chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_mech.png|thumb|right|400px|'''Figure 2. Reaction mechanisms proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2 - showing the old and the new equation). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functionality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard conditions (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite> MORE REFs needed?). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 m-1 s-1.

== Catalytic Residues and copper coordination ==

[[Image:His_brace_4ALC_label.png|thumb|left|400px|''''''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are showed as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low X-ray radiation experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial". One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure 4 shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> descibes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

[[Image:AA10diversityPNG.png|thumb|right|400px|'''''''''Figure 4. LPMO active sites.''' Active sites and their "second shell" of CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite> ]]
== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely invovled in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with the copper ion (that is essential for activity) bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as dicussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

Auxiliary Activity Family 10

2018-01-16T13:47:42Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg 2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically actove AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 bind chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_mech.png|thumb|right|400px|'''Figure 2. Reaction mechanisms proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2 - showing the old and the new equation). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functionality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard conditions (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite> MORE REFs needed?). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 m-1 s-1.

== Catalytic Residues and copper coordination ==

[[Image:His_brace_4ALC_label.png|thumb|right|400px|''''''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are showed as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low X-ray radiation experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial". One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure 4 shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> descibes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

[[Image:His_brace_4ALC_label.png|thumb|right|400px|'''''''''Figure 4. LPMO active sites.''' Active sites and their "second shell" of CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite> ]]
== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely invovled in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with the copper ion (that is essential for activity) bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as dicussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

File:AA10 diversityPNG.PNG

2018-01-16T13:27:31Z

Gustav Vaaje-Kolstad: Active site surroundings of AA10s

Active site surroundings of AA10s

Auxiliary Activity Family 10

2018-01-16T13:08:27Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg 2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically actove AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 bind chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_mech.png|thumb|right|400px|'''Figure 2. Reaction mechanisms proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2 - showing the old and the new equation). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functionality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard conditions (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite> MORE REFs needed?). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 m-1 s-1.

== Catalytic Residues and copper coordination ==[[Image:His_brace_4ALC_label.png|thumb|right|400px|''''''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are showed as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low X-ray radiation experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial". One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. X) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure Y (=Fig. 3C in <cite>Forsberg2014</cite>) shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> descibes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely invovled in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with the copper ion (that is essential for activity) bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as dicussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

Auxiliary Activity Family 10

2018-01-16T12:57:22Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg 2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically actove AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 bind chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_mech.png|thumb|right|400px|'''Figure 2. Reaction mechanisms proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2 - showing the old and the new equation). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functionality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard conditions (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite> MORE REFs needed?). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 m-1 s-1.

== Catalytic Residues and copper coordination ==[[Image:His_brace_4ALC_label.png|thumb|right|400px|''''''Figure 3. The active site histidine brace of EfLPMO10 (EfCBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom, which is in the Cu(II) state, is showed as a golden sphere. Water molecules are showed as red spheres. The figure is made from the structure of EfLPMO10 (also known as "EfCBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by low X-ray radation experiments, thus preventing X-ray photoreduction and thereby yielding an active site containing a copper atom in its Cu(II) state.]]

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. X) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial". One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. X) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure Y (=Fig. 3C in <cite>Forsberg2014</cite>) shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> descibes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely invovled in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with the copper ion (that is essential for activity) bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as dicussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

File:His brace 4ALC label.png

2018-01-16T12:49:46Z

Gustav Vaaje-Kolstad: His brace configuration of EfLPMO10A (EfCBM33) in the unoxidized state

His brace configuration of EfLPMO10A (EfCBM33) in the unoxidized state

Auxiliary Activity Family 10

2018-01-16T12:10:54Z

Gustav Vaaje-Kolstad:

{{UnderConstruction}}
* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
* [[Responsible Curator]]: ^^^Vincent Eijsink^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 10'''
|-
|'''Clan'''
|Structurally related to [[AA9]] & [[AA11]] & [[AA13]]
|-
|'''Mechanism'''
|lytic oxidase
|-
|'''Active site residues'''
|mononuclear copper ion
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}AA10.html
|}
</div>


== Substrate specificities ==
[[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from <cite>Horn2012</cite>.]]
Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg 2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from ''S. marcescens'' <cite>Vaaje-Kolstad2010</cite> and EfCBM33A from ''E. faecalis'' <cite>Vaaje-Kolstad2012 </cite> are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from ''S. coelicolor'' (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module <cite>Forsberg2011 Forsberg2014 Forsbergb2014</cite>. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically actove AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 bind chitin <cite>Wong2012 Loose2014</cite>.

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins <cite>Vaaje-Kolstad2005-1 Vaaje-Kolstad2017</cite>. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 <cite>Forsberg2018</cite>. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

== Kinetics and Mechanism ==

[[Image:Rx_mech.png|thumb|right|400px|'''Figure 2. Reaction mechanisms proposed for LPMOs in a) 2010 and b) 2017.]]

The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2 - showing the old and the new equation). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.

One challenging element of understanding how LPMOs work relates to the required delivery of two electrons to a substrate-bound enzyme whose single co-factor only allows storage of one electron. While several scenarios have been proposed, a radical and experimentally supported solution to this problem was recently proposed by Bissaro et al <cite>Bissaro2017</cite>. As shown in Fig. 2, panel b, the proposal is that hydrogen peroxide rather than molecular oxygen is the preferred co-substrate of LPMOs. The H2O2-based mechanism would only require a priming reduction of the LPMO to its Cu(I) state, which would then be capable of carrying out multiple catalytic cycles while having its electrons and oxygen delivered in the form of partially reduced O2, namely H2O2. Bissaro ''et al.'' point out that H2O2 will be generated under the reaction conditions that are normally used to assess LPMO functioality. Further work is needed to assess the validity of the various proposed LPMO mechanisms. One key challenge in such work is that LPMOs become rapidly inactivated due to oxidative damage in the catalytic center <cite>Bissaro2017 Kuusk2018</cite>

Reported catalytic rates of LPMOs, including AA10 LPMOs, under standard condiitons (= the presence of a reductant and O2), are remarkably low and tend to vary from 1 to 10 per minute (e.g. <cite>Vaaje-Kolstad2010 Agger2014 Frandsen2016</cite> MORE REFs needed?). Although not usually reported in the literature, progress curves for LPMO reactions are notoriously non-linear and there is thus little available kinetic data (see <cite>Frandsen2016 Kuusk2018</cite> for relatively high quality kinetic data). Importantly, it has been shown that LPMO rates can be drastically enhanced by using light <cite>Cannella2016</cite> or H2O2 <cite>Bissaro2017</cite> to drive the LPMO reaction. In a recent detailed kinetic study of CBP21, using hydrogen peroxide to drive the reaction, Kuusk et al <cite>Kuusk2018</cite> determined the kcat for chitin oxidation found here to be 5.6 s-1, and concluded that the kcat/Km for H2O2-driven degradation of chitin was in the order of 106 m-1 s-1.

== Catalytic Residues and copper coordination ==
Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. X) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial". One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. X) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure Y (=Fig. 3C in <cite>Forsberg2014</cite>) shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> descibes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

== Three-dimensional structures ==
In 2005 the structure CBP21 from ''S. marcescens'' was solved and represents the first structure in the AA10 family [{{PDBlink}}2bem 2BEM] <cite>Vaaje-Kolstad2005-1</cite> and the first structure of an LPMO The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site [{{PDBlink}}2ben 2BEN] <cite>Vaaje-Kolstad2005-2</cite>. The second AA10 structure, one of two AA10 from ''Burkholderia pseudomallei'' 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by Seattle Structural Genomics Center for Infectious Disease [{{PDBlink}}3uam 3UAM]. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from ''Vibrio cholerae'' O1 biovar El Tor str. N16961 [{{PDBlink}}2xwx 2XWX] <cite>,Wong2012</cite>. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely invovled in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from ''Enterococcus faecalis'' [{{PDBlink}}4a02 4A02] <cite>Vaaje-Kolstad2012</cite> was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR [{{PDBlink}}2lhs 2LHS] <cite>Aachmann2012</cite>, and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs <cite>Quinlan2011</cite>. In spring 2013 the structure of the single AA10 harbored by''Bacillus amyloliquefaciens'' DSM7 (BaCBM33) was published <cite>Hemsworth2013</cite>. The latter publication contained three structures: the apo-enzyme [{{PDBlink}}2yow 2YOW] and two structures containing a reduced copper ion bound to the active site [{{PDBlink}}2yoy 2YOY][{{PDBlink}}2yox 2YOX], thus being the first AA10 structures with the copper ion (that is essential for activity) bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 <cite>Forsberg2014</cite>. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as dicussed by Hemsworth et al <cite>Hemsworth2013-2</cite> and Gudmundsson et al <cite>Gudmundsson2014</cite>.

== Family Firsts ==
;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

== References ==
<biblio>
#Forsberg2011 pmid=21748815
#Vaaje-Kolstad2005-1 pmid=15590674
#Vaaje-Kolstad2005-2 pmid=15929981
#Vaaje-Kolstad2010 pmid=20929773
#Aachmann2012 pmid=23112164
#Vaaje-Kolstad2012 pmid=22210154
#Wong2012 pmid=22253590
#Hemsworth2013 pmid=23540833
#Fuchs1986 pmid=16347012
#Suzuki1998 pmid=9501524
#Walter2008 pmid=18438642
#Chu2001 pmid=11429457
#Saito2001 pmid=11229920
#Kolbe1998 pmid=9611804
#Zeltins1997 pmid=9208950
#Folders2000 pmid=10671445
#Sanches2011 pmid=21131525
#Hemsworth2013-2 pmid=23769965
#Horn2012 pmid=22747961
#Schnellman1994 pmid=7815940
#Forsberg2014 pmid=24912171
#Forsbergb2014 pmid=24559135
#Forsberg2018 pmid=29222333
#Vaaje-Kolstad2009 pmid=19348025
#Loose2014 pmid=25109775
#Vaaje-Kolstad2017 pmid=28086105
#Gudmundsson2014 pmid=24828494
#Quinlan2011 pmid=21876164
#Harris2010 pmid=20230050
#Frandsen2016 pmid=26928935
#Span2017 pmid=28257189
#Bacik2017 pmid=28481095
#Courtade2016 pmid=27152023
#Phillips2011 pmid=22004347
#Isaksen2014 pmid=24324265
#Beeson2015 pmid=25784051
#Walton2016 pmid=27094791
#Bissaro2017 pmid=28846668
#Bertini2018 pmid=29232119
#Meier2018 pmid=29155571
#Kuusk2018 pmid=29138240
#Agger2014 pmid=24733907
#Cannella2016 pmid=27041218
#Crouch2016 pmid=26801613
</biblio>

[[Category:Auxiliary Activity Families|AA010]]

File:Rx mech.png

2018-01-16T11:52:54Z

Gustav Vaaje-Kolstad: Reaction mech LPMOs

Reaction mech LPMOs

Auxiliary Activity Family 10

2015-12-10T08:22:47Z

Gustav Vaaje-Kolstad:

Auxiliary Activity Family 10

2015-12-10T07:59:28Z

Gustav Vaaje-Kolstad:

Auxiliary Activity Family 10

2015-12-10T07:56:55Z

Gustav Vaaje-Kolstad:

Auxiliary Activity Family 10

2015-12-10T07:50:19Z

Gustav Vaaje-Kolstad:

Auxiliary Activity Family 10

2015-12-10T07:36:01Z

Gustav Vaaje-Kolstad:

Auxiliary Activity Family 10

2015-12-10T07:32:15Z

Gustav Vaaje-Kolstad:

Auxiliary Activity Family 10

2015-12-10T07:22:38Z

Gustav Vaaje-Kolstad:

Auxiliary Activity Family 10

2015-12-10T07:22:08Z

Gustav Vaaje-Kolstad:

Auxiliary Activity Family 10

2013-08-01T08:56:05Z

Gustav Vaaje-Kolstad:

Auxiliary Activity Family 10

2013-08-01T08:54:58Z

Gustav Vaaje-Kolstad:

Auxiliary Activity Family 10

2013-08-01T08:48:12Z

Gustav Vaaje-Kolstad:

Auxiliary Activity Family 10

2013-08-01T08:44:33Z

Gustav Vaaje-Kolstad:

File:CBP21 binding chitin modeled.jpg

2013-08-01T08:43:33Z

Gustav Vaaje-Kolstad: