https://www.cazypedia.org/api.php?action=feedcontributions&feedformat=atom&user=Glyn+HemsworthCAZypedia - User contributions [en-ca]2026-05-04T18:06:18ZUser contributionsMediaWiki 1.35.10https://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13558Auxiliary Activity Family 132019-02-19T09:33:19Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}}<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on starch <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate. Despite significant efforts, it has not been possible to determine an oligosaccharide bound structure for an AA13 which would be particularly beneficial to delineating the adaptions necessary to allow activity on an &alpha;-1,4-linked substrate <cite>Frandsen2017</cite>.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemsworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Frandsen2017 pmid=28045386<br />
<br />
#Hemsworth2013 pmid=23769965<br />
<br />
#Hemsworth2014 pmid=24362702<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13557Auxiliary Activity Family 132019-02-18T09:57:55Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}}<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on starch <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate. Despite significant efforts, it has not been possible to determine an oligosaccharide bound structure for an AA13 which would be particularly beneficial to delineating the adaptions necessary to allow activity on an &alpha;-1,4-linked substrate <cite>Frandsen2017</cite>.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Frandsen2017 pmid=28045386<br />
<br />
#Hemsworth2013 pmid=23769965<br />
<br />
#Hemsworth2014 pmid=24362702<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13556Auxiliary Activity Family 132019-02-18T09:54:49Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}}<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on starch <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate. Despite significant efforts, it has not been possible to determine an oligosaccharide bound structure for an AA13 which would be particularly beneficial to delineating the adaptions necessary to allow activity on an &alpha;-1,4-linked substrate <cite>Frandsen2017</cite>.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Frandsen2017 pmid=28045386<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13555Auxiliary Activity Family 132019-02-18T09:50:25Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}}<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on starch <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate. Despite significant efforts, it has not been possible to determine an oligosaccharide bound structure for an AA13 which would be particularly beneficial to delineating the adaptions necessary to allow activity on an &alpha;-1,4-linked substrate <cite>Frandsen2017</cite>.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
#Frandsen2017 pmid=28045386<br />
#Frandsen2018 pmid=26928935<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13554Auxiliary Activity Family 132019-02-14T13:50:58Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}}<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on starch <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate. Despite significant efforts, it has not been possible to determine an oligosaccharide bound structure for an AA13 which would be particularly beneficial to delineating the adaptions necessary to allow activity on an &alpha;-1,4-linked substrate <cite>Frandsen2017</cite>.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was indicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch and may imply that AA13s are not subject to the substrate controlled activation mechanism suggested for [[AA9]]s <cite>Frandsen2018</cite>.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
#Frandsen2017 pmid=28045386<br />
#Frandsen2018 pmid=26928935<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13553Auxiliary Activity Family 132019-02-14T13:49:11Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}}<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on starch <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate. Despite significant efforts, it has not been possible to determine an oligosaccharide bound structure for an AA13 which would be particularly beneficial to delineating the adaptions necessary to allow activity on an &alpha;-1,4-linked substrate <cite>Frandsen2017</cite>.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch and may imply that AA13s are not subject to the substrate controlled activation mechanism suggested for [[AA9]]s <cite>Frandsen2018</cite>.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
#Frandsen2017 pmid=28045386<br />
#Frandsen2018 pmid=26928935<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13552Auxiliary Activity Family 132019-02-14T13:32:04Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}}<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on starch <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate. Despite significant efforts, it has not been possible to determine an oligosaccharide bound structure for an AA13 which would be particularly beneficial to delineating the adaptions necessary to allow activity on an &alpha;-1,4-linked substrate <cite>Frandsen2017</cite>.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch and may imply that AA13s are not subject to the substrate controlled activation mechanism suggested for [[AA9]]s <cite>Frandsen2018</cite><br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
#Frandsen2017 pmid=28045386<br />
#Frandsen2018 pmid=26928935<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13551Auxiliary Activity Family 132019-02-14T13:25:54Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on starch <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate. Despite significant efforts, it has not been possible to determine an oligosaccharide bound structure for an AA13 which would be particularly beneficial to delineating the adaptions necessary to allow activity on an &alpha;-1,4-linked substrate <cite>Frandsen2017</cite>. <br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch and may imply that AA13s are not subject to the substrate controlled activation mechanism suggested for [[AA9]]s <cite>Frandsen2018</cite><br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
#Frandsen2017 pmid=28045386<br />
#Frandsen2018 pmid=26928935<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13550Auxiliary Activity Family 132019-02-14T12:52:27Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on starch <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate. Despite significant efforts, it has not been possible to determine an oligosaccharide bound structure for an AA13 which would be particularly beneficial to delineating the adaptions necessary to allow activity on an &alpha;-1,4-linked substrate <cite>Frandsen2017</cite>. <br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch and may imply that AA13s are not subject to the substrate controlled activation mechanism suggested for [[AA9]]s <cite>Frandsen2018</cite><br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
#Frandsen2017 pmid=28045386<br />
#Frandsen2018 pmid=26928935<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13549Auxiliary Activity Family 132019-02-14T12:50:34Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate. Despite significant efforts, it has not been possible to determine an oligosaccharide bound structure for an AA13 which would be particularly beneficial to delineating the adaptions necessary to allow activity on an &alpha;-1,4-linked substrate <cite>Frandsen2017</cite>. <br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch and may imply that AA13s are not subject to the substrate controlled activation mechanism suggested for [[AA9]]s <cite>Frandsen2018</cite><br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
#Frandsen2017 pmid=28045386<br />
#Frandsen2018 pmid=26928935<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13548Auxiliary Activity Family 132019-02-14T12:49:49Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate. Despite significant efforts, it has not been possible to determine an oligosaccharide bound structure for an AA13 which would be particularly beneficial to delineating the adaptions necessary to allow activity on an &alpha;-1,4-linked substrate. <br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch and may imply that AA13s are not subject to the substrate controlled activation mechanism suggested for [[AA9]]s <cite>Frandsen2018</cite><br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
#Frandsen2017 pmid=28045386<br />
#Frandsen2018 pmid=26928935<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13547Auxiliary Activity Family 132019-02-14T12:43:11Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch and may imply that AA13s are not subject to the substrate controlled activation mechanism suggested for [[AA9]]s <cite>Frandsen2018</cite><br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
#Frandsen2018 pmid=26928935<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13546Auxiliary Activity Family 132019-02-14T12:41:50Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch and may imply that AA13s are not subject to the substrate controlled activation mechanism suggested for [[AA9]]s <cite>Frandsen2018</cite><br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13545Auxiliary Activity Family 132019-02-14T12:38:40Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy with a &beta;-amylase during degradation assays using retrograded starch as the substrate. The combined action of these enzymes resulted in >100-fold increase in the release of maltose compared to &beta;-amylase acting alone, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) also represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. In addition, the accessibility of this axial position to solvent has been implicated in mediating the regiospecificity of [[AA10]]s and [[AA9]]s, with secondary sphere resides around the copper becoming increasingly studied as well <cite>Forsberg2014 Borisova2015 Span2017 Forsberg2017</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13544Auxiliary Activity Family 132019-02-14T12:34:19Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation ([{{PatentLink}}WO2014197705A1 WO2014197705A1]).<br />
<br />
Of the AA13s that have been biochemically characterised to date, activity has only been demonstrate on starch and related substrates <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy in starch degradation assays in combination with a &beta;-amylase, boosting the production of maltose by this enzyme >100-fold, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) also represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. In addition, the accessibility of this axial position to solvent has been implicated in mediating the regiospecificity of [[AA10]]s and [[AA9]]s, with secondary sphere resides around the copper becoming increasingly studied as well <cite>Forsberg2014 Borisova2015 Span2017 Forsberg2017</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13543Auxiliary Activity Family 132019-02-14T12:31:20Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation [{{PatentLink}}WO2014197705A1 WO2014197705A1].<br />
<br />
All AA13s that have been biochemically characterised to date are active on starch <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Density Functional Theory (DFT) calculations have been performed using [[AA9]] structures as a starting point leading to the currently favoured mechanism being a Cu(II)-oxyl based rebound mechanism <cite>Kim2014 Bertini2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy in starch degradation assays in combination with a &beta;-amylase, boosting the production of maltose by this enzyme >100-fold, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) also represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. In addition, the accessibility of this axial position to solvent has been implicated in mediating the regiospecificity of [[AA10]]s and [[AA9]]s, with secondary sphere resides around the copper becoming increasingly studied as well <cite>Forsberg2014 Borisova2015 Span2017 Forsberg2017</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13542Auxiliary Activity Family 132019-02-14T12:05:18Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. Prior to this, genes now known to code for AA13s were demonstrated to produce proteins capable of boosting &alpha; and &beta;-amylase activity during starch, amylose and amylopectin degradation [{{PatentLink}}WO2014197705A1 WO2014197705A1] While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach.<br />
<br />
All AA13s that have been biochemically characterised to date are active on starch <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Density Functional Theory (DFT) calculations have been performed using [[AA9]] structures as a starting point leading to the currently favoured mechanism being a Cu(II)-oxyl based rebound mechanism <cite>Kim2014 Bertini2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy in starch degradation assays in combination with a &beta;-amylase, boosting the production of maltose by this enzyme >100-fold, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) also represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. In addition, the accessibility of this axial position to solvent has been implicated in mediating the regiospecificity of [[AA10]]s and [[AA9]]s, with secondary sphere resides around the copper becoming increasingly studied as well <cite>Forsberg2014 Borisova2015 Span2017 Forsberg2017</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=13541Auxiliary Activity Family 132019-02-14T11:50:08Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised in the academic literature was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach.<br />
<br />
All AA13s that have been biochemically characterised to date are active on starch <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Density Functional Theory (DFT) calculations have been performed using [[AA9]] structures as a starting point leading to the currently favoured mechanism being a Cu(II)-oxyl based rebound mechanism <cite>Kim2014 Bertini2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy in starch degradation assays in combination with a &beta;-amylase, boosting the production of maltose by this enzyme >100-fold, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) also represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. In addition, the accessibility of this axial position to solvent has been implicated in mediating the regiospecificity of [[AA10]]s and [[AA9]]s, with secondary sphere resides around the copper becoming increasingly studied as well <cite>Forsberg2014 Borisova2015 Span2017 Forsberg2017</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=11914Auxiliary Activity Family 132018-01-05T10:00:22Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach.<br />
<br />
All AA13s that have been biochemically characterised to date are active on starch <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Density Functional Theory (DFT) calculations have been performed using [[AA9]] structures as a starting point leading to the currently favoured mechanism being a Cu(II)-oxyl based rebound mechanism <cite>Kim2014 Bertini2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy in starch degradation assays in combination with a &beta;-amylase, boosting the production of maltose by this enzyme >100-fold, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) also represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. In addition, the accessibility of this axial position to solvent has been implicated in mediating the regiospecificity of [[AA10]]s and [[AA9]]s, with secondary sphere resides around the copper becoming increasingly studied as well <cite>Forsberg2014 Borisova2015 Span2017 Forsberg2017</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. crassa</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=11913Auxiliary Activity Family 132018-01-05T09:59:57Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach.<br />
<br />
All AA13s that have been biochemically characterised to date are active on starch <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Density Functional Theory (DFT) calculations have been performed using [[AA9]] structures as a starting point leading to the currently favoured mechanism being a Cu(II)-oxyl based rebound mechanism <cite>Kim2014 Bertini2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy in starch degradation assays in combination with a &beta;-amylase, boosting the production of maltose by this enzyme >100-fold, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) also represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. In addition, the accessibility of this axial position to solvent has been implicated in mediating the regiospecificity of [[AA10]]s and [[AA9]]s, with secondary sphere resides around the copper becoming increasingly studied as well <cite>Forsberg2014 Borisova2015 Span2017 Forsberg2017</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>N. Crass</i> <cite>Vu2014</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=11912Auxiliary Activity Family 132018-01-04T22:55:59Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach.<br />
<br />
All AA13s that have been biochemically characterised to date are active on starch <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Density Functional Theory (DFT) calculations have been performed using [[AA9]] structures as a starting point leading to the currently favoured mechanism being a Cu(II)-oxyl based rebound mechanism <cite>Kim2014 Bertini2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy in starch degradation assays in combination with a &beta;-amylase, boosting the production of maltose by this enzyme >100-fold, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) also represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side (Figure 2, top) <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. In addition, the accessibility of this axial position to solvent has been implicated in mediating the regiospecificity of [[AA10]]s and [[AA9]]s, with secondary sphere resides around the copper becoming increasingly studied as well <cite>Forsberg2014 Borisova2015 Span2017 Forsberg2017</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum (Figure 2, bottom) <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>A. oryzae</i> <cite>LoLeggio2015</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=11911Auxiliary Activity Family 132018-01-04T22:54:11Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach.<br />
<br />
All AA13s that have been biochemically characterised to date are active on starch <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Density Functional Theory (DFT) calculations have been performed using [[AA9]] structures as a starting point leading to the currently favoured mechanism being a Cu(II)-oxyl based rebound mechanism <cite>Kim2014 Bertini2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy in starch degradation assays in combination with a &beta;-amylase, boosting the production of maltose by this enzyme >100-fold, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) also represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. In addition, the accessibility of this axial position to solvent has been implicated in mediating the regiospecificity of [[AA10]]s and [[AA9]]s, with secondary sphere resides around the copper becoming increasingly studied as well <cite>Forsberg2014 Borisova2015 Span2017 Forsberg2017</cite>. An interesting feature of the electron paramagnetic resonance (EPR) spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>A. oryzae</i> <cite>LoLeggio2015</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_13&diff=11910Auxiliary Activity Family 132018-01-04T22:52:38Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Glyn Hemsworth^^^ & ^^^Leila LoLeggio^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 13'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]], [[AA10]] & [[AA11]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion coordinated by the “histidine brace”<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA13.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA13 family represents the fourth family of Lytic Polysaccharide Monooxygenases (LPMOs) that has been identified. Found in fungi, the first member of this family to be identified and characterised was isolated from <i>Neurospora crassa</i> by Vu et al <cite>Vu2014</cite>. While searching the <i>Neurospora crassa</i> genome for new putative LPMO sequences, they identified a sequence that appeared to code for an LPMO with a C-terminal [[CBM20]] domain but lacked significant overall sequence similarity to other [[AA9]], [[AA10]] or [[AA11]] LPMOs. Not long after Lo Leggio et al <cite>LoLeggio2015</cite> also identified and characterised AA13 family members from <i>Aspergilllus nidulans</i> and <i>Aspergillus oryzae</i> using a similar approach.<br />
<br />
All AA13s that have been biochemically characterised to date are active on starch <cite>Vu2014 LoLeggio2015</cite>. The expression of these enzymes has also been shown to be heavily unregulated during growth of <i>A. nidulans</i> on this substrate <cite>Nekiunaite2016a</cite>. As for other LPMOs, AA13s utilise copper and an electron donor to oxidatively introduce chain breaks into the &alpha;-1,4-linked glucose polymers that form starch <cite>Vu2014 LoLeggio2015</cite>. AA13s specifically attack at the C1 position of the sugar ring forming oligosaccharide products with lactones at the reducing end which are then additionally hydrated to form aldonic acid terminated maltodextrins <cite>LoLeggio2015</cite>. The C1 specificity suggests that these LPMOs should be able to oxidatively attack both the &alpha;-1,4- and &alpha;-1,6-linkages found in amylopectin <cite>Vu2015</cite>. Interestingly, AA13s are currently the only LPMOs characterised to date that act on a glucan polymer formed from anything other than &beta;-1,4-linkages <cite>Vu2016</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
All LPMOs ([[AA9]], [[AA10]], [[AA11]], & AA13) are copper dependent monooxygenases and there is considerable work ongoing in trying to elucidate their detailed reaction mechanism <cite>Walton2017</cite>. Density Functional Theory (DFT) calculations have been performed using [[AA9]] structures as a starting point leading to the currently favoured mechanism being a Cu(II)-oxyl based rebound mechanism <cite>Kim2014 Bertini2017</cite>. Starch, being an &alpha;-1,4-linked substrate, poses a different challenge for AA13s to be able to oxidatively attack at the C1 carbon. Whether there is a significant difference in mechanism required in order to oxidise starch is currently unclear.<br />
<br />
[[File:AA13_Cazypedia_Small.gif|thumb|right|450x356px|'''Figure 1. Structure of AA13 from <i>Aspergillus oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' In the above animation the structure is shown initially in cartoon representation coloured by secondary structure and the transparent surface shown in grey. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. As the animation proceeds the surface surrounding the active site is shown to illustrate the presence of a groove that passes over the active site. This groove is thought to accommodate the helical structure of an &alpha;-1,4-linked amylopectin substrate.]]<br />
<br />
Performing kinetic measurements on LPMOs has been notoriously difficult. Several key aspects of AA13 biochemistry have been observed however. The <i>A. nidulans</i> AA13 showed significant synergy in starch degradation assays in combination with a &beta;-amylase, boosting the production of maltose by this enzyme >100-fold, representing one of the most significant boosting activities observed so far for any LPMO <cite>LoLeggio2015</cite>. Also highlighted in this study was the importance of the reducing agent used to activate the enzyme with cysteine providing greater activity than ascorbate which is typically used to activate LPMOs. Vu et al <cite>Vu2014</cite> also showed that cellobiose dehydrogenase (CDH composed of [[AA3]] and [[AA8]] domains) also represents a good activating partner for AA13s as has been observed for [[AA9]]s. The importance of the [[CBM20]] module for AA13 activity has also been highlighted. The <i>A. oryzae</i> AA13, which naturally lacks a CBM, did not appear to be active in assays on starch while the [[CBM20]] appended A.nidulans enzyme showed clear activity <cite>LoLeggio2015</cite>. Indeed the presence of the [[CBM20]] domain has been demonstrated to confer typical starch and &beta;-cyclodextrin binding properties to AA13s and is suggested to mediate most of the substrate binding properties of this family of enzymes <cite>Nekiunaite2017b</cite>.<br />
<br />
[[File:AA13_Active_Site.png|thumb|right|331x654px|'''Figure 2. The active site architecture observed for the AA13 from <i>A. oryzae</i> (PDB ID [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite>).''' '''Top:''' the active site residues surrounding the copper ion are shown. The Cu is shown as a sphere and is in the Cu(I) state due to photo reduction from the X-ray beam. '''Bottom:''' EPR spectrum observed for the AA13 from <i>A. oryzae</i> revealing super hyperfine coupling. The raw data are shown in black with the simulated data in red.]]<br />
<br />
== Three-dimensional structures ==<br />
The core fold of the AA13 structure is similar to all other LPMOs ([[AA9]], [[AA10]], & [[AA11]]) representing a &beta;-sandwich immunoglobulin like fold <cite>LoLeggio2015</cite>. There are some significant structural differences compared to the other families however. Firstly, the AA13 structure reveals additional helical secondary structure elements that are not found in other LPMOs (Figure 1). The most prominent difference however is surrounding the copper active site. Where in other LPMOs, the copper histidine brace is found at the centre of a flat or slightly concave surface ([[AA9]], [[AA10]], & [[AA11]]), a distinct groove can be observed passing over the active site of AA13 (Figure 1). This is an adaptation that is thought to be necessary to accommodate the helical structure of an amylopectin substrate.<br />
<br />
== Catalytic Residues ==<br />
The “histidine brace” motif is used to bind the active site copper in AA13 <cite>LoLeggio2015</cite>, as it is for all other LPMOs studied to date ([[AA9]], [[AA10]] & [[AA11]]) <cite>Vaaje-Kolstad2017</cite>. This motif sees the amino group and imidazole side chain of the N-terminal histidine, together with the imidazole group of a second histidine, directly coordinate the copper ion in a T-shaped geometry. For AA13s, one axial position is occupied by a tyrosine on one side of the copper ion, with a loop containing a glycine approaching near to the copper on the other side <cite>LoLeggio2015</cite>. In [[AA10]]s and [[AA11]]s alanine in this position has been suggested as a possible important factor in distorting the arrangement of water molecules around the active site which may have mechanistic consequences in these families <cite>Hemworth2013 Hemsworth2014</cite>. In addition, the accessibility of this axial position to solvent has been implicated in mediating the regiospecificity of [[AA10]]s and [[AA9]]s, with secondary sphere resides around the copper becoming increasingly studied as well <cite>Forsberg2014 Borisova2015 Span2017 Forsberg2017</cite>. An interesting feature of the electron paramagnetic spectrum observed for the <i>A. nidulans</i> and <i>A. oryzae</i> AA13s was the presence of superhyperfine coupling in the spectrum <cite>LoLeggio2015</cite>. This was inidicative of a potentially more ordered active site arrangement around the copper which may be the result of structural changes that are required in order to oxidise starch.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA13 from <i>A. oryzae</i> <cite>LoLeggio2015</cite>.<br />
;First demonstration of oxidative cleavage: The <i>N. crassa</i> AA13 was shown to produce oxidised malto-oligosaccharides in the presence of oxygen and reducing agents <cite>Vu2014</cite>.<br />
;First 3-D structure: AA13 from ''A. oryzae'' with Cu+ [{{PDBlink}}4opb 4OPB] <cite>LoLeggio2015</cite><br />
<br />
== References ==<br />
<biblio><br />
#Vu2014 pmid=25201969<br />
#LoLeggio2015 pmid=25608804<br />
#Nekiunaite2016a Nekiunaite, L., Arntzen, M.Ø., Svensson, B., Vaaje-Kolstad, G., Abou Hachem, M. (2016) Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by <i>Aspergillus nidulans</i> grown on different starches. Biotechnology for Biofuels. 9, 187 [http://dx.doi.org/10.1186/s13068-016-0604-0]<br />
#Vu2016 pmid=27170366<br />
#Walton2017 pmid=27094791<br />
#Bertini2017 pmid=29232119<br />
#Kim2014 pmid=24344312<br />
#Nekiunaite2017b pmid=27397613<br />
#Vaaje-Kolstad2017 pmid=28086105<br />
#Forsberg2014 pmid=24912171<br />
#Borisova2015 pmid=26178376<br />
#Span2017 pmid=28257189<br />
#Forsberg2017 pmid=29222333<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA13]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=File:AA13_Active_Site.png&diff=11909File:AA13 Active Site.png2018-01-04T16:36:42Z<p>Glyn Hemsworth: </p>
<hr />
<div></div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=File:AA13_Cazypedia_Small.gif&diff=11908File:AA13 Cazypedia Small.gif2018-01-04T16:04:51Z<p>Glyn Hemsworth: </p>
<hr />
<div></div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=User:Glyn_Hemsworth&diff=11907User:Glyn Hemsworth2018-01-04T15:57:51Z<p>Glyn Hemsworth: </p>
<hr />
<div><br />
[[File:GlynHemsworth.jpg|200px|right]]<br />
Glyn Hemsworth obtained his B.Sc. in Biochemistry from the University of Sheffield where he stayed on to complete his PhD under the supervision of Prof. Peter Artymiuk, primarily focussing on the structural characterisation of an unusual Flap Endonuclease homologue from ''Escherichia coli''. In 2009 he moved to the Structural Biology Laboratory at the University of York where he initially worked on dUTPases from ''Trypanosoma'' and ''Leishmania'' species with Prof Keith Wilson. In 2012 he moved into the carbohydrate field taking up a post-doctoral position with Prof ^^^Gideon Davies^^^ where his major contributions have been in the structural and functional characterisation of lytic polysaccharide mono-oxygenases (LPMOs) <cite>Hemsworth2013</cite> in families [[AA10]] and [[AA11]], with additional contributions to characterisation of [[AA13]] as well. During his time in York he also contributed to the structural determination of several Glycoside Hydrolases from the human gut symbiont <i>Bacteroides ovatus</i>. He is now a BBSRC David Phillips and University Academic Fellow at the University of Leeds where his research is focussed on the discovery and characterisation of novel proteins that may play roles in the oxidative degradation of biomass. He has determined the crystal structures of<br />
<br />
* [[AA10]] from ''Bacillus amyloliquefaciens'' <cite>Hemsworth2013a</cite><br />
* [[AA11]] from ''Aspergillus oryzae'' <cite>Hemsworth2013b</cite><br />
* [[GH5]] from ''Bacteroides ovatus'' <cite>Larsbrink2014</cite><br />
* [[GH3]]A from ''Bacteroides ovatus'' <cite>Hemsworth2016</cite><br />
* [[GH43]]B from ''Bacteroides ovatus'' <cite>Hemsworth2016</cite><br />
* [[GH16]] from ''Bacteroides ovatus'' <cite>Tamura2017</cite><br />
<br />
<br />
----<br />
<br />
<biblio><br />
#Hemsworth2013 pmid=23769965<br />
#Hemsworth2013a pmid=23540833<br />
#Hemsworth2013b pmid=24362702<br />
#Larsbrink2014 pmid=24463512<br />
#Hemsworth2016 pmid=27466444<br />
#Tamura2017 pmid=29020628<br />
</biblio><br />
<br />
[[Category:Contributors|Hemsworth,Glyn]]<br />
<!-- ATTENTION: Make sure to replace "Lastname,Firstname" with your own name, for proper sorting of the Contributors page. --></div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=User:Glyn_Hemsworth&diff=9878User:Glyn Hemsworth2014-03-03T13:08:27Z<p>Glyn Hemsworth: </p>
<hr />
<div><br />
[[File:GlynHemsworth.jpg|200px|right]]<br />
Glyn Hemsworth obtained his B.Sc. in Biochemistry from the University of Sheffield where he stayed on to complete his PhD under the supervision of Prof. Peter Artymiuk, primarily focussing on the structural characterisation of an unusual Flap Endonuclease homologue from ''Escherichia coli''. In 2009 he moved to the Structural Biology Laboratory at the University of York where he initially worked on dUTPases from ''Trypanosoma'' and ''Leishmania'' species with Prof Keith Wilson. In 2012 he moved into the carbohydrate field taking up his current post-doctoral position with Prof ^^^Gideon Davies^^^ where his major contributions have been in the structural and functional characterisation of lytic polysaccharide mono-oxygenases (LPMOs) <cite>Hemsworth2013</cite> in families [[AA10]] and the newly discovered [[AA11]]. He has determined the crystal structures of<br />
<br />
* [[AA10]] from ''Bacillus amyloliquefaciens'' <cite>Hemsworth2013a</cite><br />
* [[AA11]] from ''Aspergillus oryzae'' <cite>Hemsworth2013b</cite><br />
* [[GH5]] from ''Bacteroides ovatus'' <cite>Larsbrink2014</cite><br />
<br />
<br />
----<br />
<br />
<biblio><br />
#Hemsworth2013 pmid=23769965<br />
#Hemsworth2013a pmid=23540833<br />
#Hemsworth2013b pmid=24362702<br />
#Larsbrink2013b pmid=24463512<br />
</biblio><br />
<br />
[[Category:Contributors|Hemsworth,Glyn]]<br />
<!-- ATTENTION: Make sure to replace "Lastname,Firstname" with your own name, for proper sorting of the Contributors page. --></div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_11&diff=9856Auxiliary Activity Family 112014-02-05T20:32:02Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Gideon Davies^^^ & ^^^Glyn Hemsworth^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 11'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]] & [[AA10]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA11.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA11 family of Lytic Polysaccharide Mono-oxygenases (LPMOs) was identified using a bioinformatic 'module walking' approach <cite>Hemsworth2013</cite>. Many [[AA9]] proteins have C-terminal domains, which are often cellulose binding CBMs (eg. [[CBM1]]), but some have no known function (also see <cite>Horn2012</cite>). Sequence searches using one of these domains (termed X278) returned hits where the domain was fused with [[GH18]] family members and another set of domains of unknown function. Closer examination of these domains revealed that they had a likely signal peptide followed by a histidine, typical of LPMOs, though they otherwise lacked significant sequence similarity to either [[AA9]] or [[AA10]] family members.<br />
<br />
The only AA11 to be isolated to to date is from ''Aspergillus oryzae'' which showed copper dependent oxidase activity on chitin in the presence of ascorbate. The observed products were a mixture of mainly aldonic acids and unmodified oligosaccharides with a small amount of a -2Da species, which may be C4 oxidation or the C1 lactone prior to ring opening. It is unknown at this stage whether AA11 family members will show any activity towards other substrates, whether they oxidise at positions other than C1 on the sugar ring or what the role of the X278 domain often found at the C-terminus is.<br />
<br />
== Kinetics and Mechanism ==<br />
AA11s, like [[AA9]]s and [[AA10]]s, are copper dependent mono-oxygenases but the chemical mechanism by which these enzymes perform the reaction is yet to be elucidated. Recent quantum mechanical simulations suggest that [[AA9]]s are likely to oxidise cellulose using a copper-oxyl, oxygen rebound mechanism <cite>Kim2013</cite> but further work is needed in this area. The differences in the copper coordination geometries between the families might further hint that they might use different mechanisms (Reviewed in <cite>Hemsworth2013c</cite>). As is the case for [[AA10]]s, the natural electron donor for AA11s is unknown, with no equivalent of cellobiose dehydrogenase yet discovered that is active on chitobiose. It is also unknown whether the N-terminal histidine in AA11s is N-methylated as is seen in [[AA9]]s and what effect this will have on the reaction performed by the enzyme. <br />
<br />
== Catalytic Residues ==<br />
Like [[AA9]] and [[AA10]], AA11 utilises copper, which it binds through the "histidine brace" consisting of the N-terminal histidine's amino and imidazole groups together with the imidazole sidechain of another histidine. It is unknown at this stage whether AA11s possess the methylated N-terminal histidine observed in [[AA9]]s as the protein was produced recombinantly in ''E. coli'' which does not carry out this modification. Like [[AA9]], a tyrosine sidechain is in close proximity to the copper ion but it does not directly coordinate the metal in this case. [[AA9]]s typically leave the opposite side to the tyrosine open to solvent where water molecules are observed coordinating the metal ion <cite>Harris2010 Karkehabadi2008 Li2012 Quinlan2011</cite>. In [[AA10]]s there is an alanine in this position, which causes a distortion in the copper coordination geometry away from octahedral <cite>Hemsworth2013b Vaaje-Kolstad2012 Vaaje-Kolstad2005 Aachmann2012</cite>. Interestingly AA11 has an alanine in a very similar position giving an active site structure somewhere between that of [[AA9]]s and [[AA10]]s. The structure was only determined with Cu(I) in the active site so the coordination geometry of Cu(II) was not directly observed but electron paramagnetic resonance spectroscopy confirmed that the copper coordination geometry lies somewhere between that of [[AA9]]s and [[AA10]]s <cite>Hemsworth2013</cite>.<br />
<br />
== Three-dimensional structures ==<br />
[[Image:AoAA11_Cazypedia.png|thumb|right|486x332px|'''Figure 1. Structure of AA11 from ''Aspergillus oryzae'' (PDB ID [{{PDBlink}}4mai 4MAI] <cite>Hemsworth2013</cite>).''' On the left the overall structure is shown in cartoon representation with the surface of the protein shown in gray. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. On the right; at the top is a view of the active site showing the coordinatiuon of the copper ion by the “histidine brace”. Below are shown the active sites of ''Thermoascus aurantiacus'' [[AA9]] (PDB ID [{{PDBlink}}2yet 2YET] <cite>Quinlan2011</cite>) and ''Bacillus amyloliquefaciens'' [[AA10]] (PDB ID [{{PDBlink}}2yoy 2YOY] <cite>Hemsworth2013b</cite>) in the same orientation showing how the AA11 forms a hybrid active site between the two families.]]<br />
<br />
Though AA11 does not share significant sequence similarity to [[AA9]] and [[AA10]] proteins, the core fold of the protein is remarkably similar forming a typical ß-sandwich immunoglobulin like fold <cite>Hemsworth2013</cite>. The active site is at the centre of a slightly concave surface which, consistent with observations in [[AA10]]s <cite>Aachmann2012 Vaaje-Kolstad2012</cite>, has very few aromatic residues suggesting that it also primarily interacts with chitin via H-bonding interactions. Given that LPMOs are oxidoreductases there is increasing interest in the role of electron transport chains within these proteins <cite>Li2012</cite>. Internal tyrosine and tryptophan residues have been implicated in these roles in [[AA9]]s <cite>Li2012</cite> and [[AA10]]s <cite>Hemsworth2013c</cite> respectively. Similarly in AA11s it is possible to trace a path through the core of the protein using tryptophans, methionine and other hydrophilic residues to the distal face of the enzyme from the active site suggesting that there may be a similar electron transport path in AA11s though these are yet to be experimentally verified in LPMOs.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA11 from ''A. oryzae'' <cite>Hemsworth2013</cite>.<br />
;First demonstration of oxidative cleavage: ''Ao''AA11 was shown to oxidatively cleave squid pen chitin producing a mixture of aldonic acids, unmodified oligosaccharides and undistinguishable lactone/C4 oxidation products <cite>Hemsworth2013</cite>.<br />
;First 3-D structure: AA11 from ''A. oryzae'' with Zn2+ [{{PDBlink}}4mah 4MAH] & Cu(I) [{{PDBlink}}4mai 4MAI] <cite>Hemsworth2013</cite><br />
<br />
== References ==<br />
<biblio><br />
#Hemsworth2013 pmid=24362702<br />
#Horn2012 pmid=22747961<br />
#Kim2013 pmid=24344312<br />
#Hemsworth2013c pmid=23769965<br />
#Harris2010 pmid=20230050<br />
#Karkehabadi2008 pmid=18723026<br />
#Li2012 pmid=22578542<br />
#Quinlan2011 pmid=21876164<br />
#Hemsworth2013b pmid=23540833<br />
#Aachmann2012 pmid=23112164<br />
#Vaaje-Kolstad2012 pmid=22210154<br />
#Vaaje-Kolstad2005 pmid=15590674<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA11]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_11&diff=9855Auxiliary Activity Family 112014-01-20T16:52:30Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Gideon Davies^^^ & ^^^Glyn Hemsworth^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 11'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]] & [[AA10]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA11.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA11 family of Lytic Polysaccharide Mono-oxygenases (LPMOs) was identified using a bioinformatic 'module walking' approach <cite>Hemsworth2013</cite>. Many [[AA9]] proteins have C-terminal domains, which are often cellulose binding CBMs (eg. [[CBM1]]), but some have no known function (also see <cite>Horn2012</cite>). Sequence searches using one of these domains (termed X278) returned hits where the domain was fused with [[GH18]] family members and another set of domains of unknown function. Closer examination of these domains revealed that they had a likely signal peptide followed by a histidine, typical of LPMOs, though they otherwise lacked significant sequence similarity to either [[AA9]] or [[AA10]] family members.<br />
<br />
The only AA11 to be isolated to to date is from ''Aspergillus oryzae'' which showed copper dependent oxidase activity on chitin in the presence of ascorbate. The observed products were a mixture of mainly aldonic acids and unmodified oligosaccharides with a small amount of a -2Da species, which may be C4 oxidation or the C1 lactone prior to ring opening. It is unknown at this stage whether AA11 family members will show any activity towards other substrates, whether they oxidise at positions other than C1 on the sugar ring or what the role of the X278 domain often found at the C-terminus is.<br />
<br />
== Kinetics and Mechanism ==<br />
AA11s, like [[AA9]]s and [[AA10]]s, are copper dependent mono-oxygenases but the chemical mechanism by which these enzymes perform the reaction is yet to be elucidated. Recent quantum mechanical simulations suggest that [[AA9]]s are likely to oxidise cellulose using a copper-oxyl, oxygen rebound mechanism <cite>Kim2013</cite> but further work is needed in this area. The differences in the copper coordination geometries between the families might further hint that they might use different mechanisms (Reviewed in <cite>Hemsworth2013c</cite>). As is the case for [[AA10]]s, the natural electron donor for AA11s is unknown, with no equivalent of cellobiose dehydrogenase yet discovered that is active on chitobiose. It is also unknown whether the N-terminal histidine in AA11s is N-methylated as is seen in [[AA9]]s and what effect this will have on the reaction performed by the enzyme. <br />
<br />
== Catalytic Residues ==<br />
Like [[AA9]] and [[AA10]], AA11 utilises copper, which it binds through the "histidine brace" consisting of the N-terminal histidine's amino and imidazole groups together with the imidazole sidechain of another histidine. It is unknown at this stage whether AA11s possess the methylated N-terminal histidine observed in [[AA9]]s as the protein was produced recombinantly in ''E. coli'' which does not carry out this modification. Like [[AA9]], a tyrosine sidechain is in close proximity to the copper ion but it does not directly coordinate the metal in this case. [[AA9]]s typically leave the opposite side to the tyrosine open to solvent where water molecules are observed coordinating the metal ion <cite>Harris2010 Karkehabadi2008 Li2012 Quinlan2011</cite>. In [[AA10]]s there is an alanine in this position, which causes a distortion in the copper coordination geometry away from octahedral <cite>Hemsworth2013b Vaaje-Kolstad2012 Vaaje-Kolstad2005 Aachmann2012</cite>. Interestingly AA11 has an alanine in a very similar position giving an active site structure somewhere between that of [[AA9]]s and [[AA10]]s. The structure was only determined with Cu(I) in the active site so the coordination geometry of Cu(II) was not directly observed but electron paramagnetic resonance spectroscopy confirmed that the copper coordination geometry lies somewhere between that of [[AA9]]s and [[AA10]]s <cite>Hemsworth2013</cite>.<br />
<br />
== Three-dimensional structures ==<br />
[[Image:AoAA11_Cazypedia.png|thumb|right|486x332px|'''Figure 1. Structure of AA11 from ''Aspergillus oryzae'' (PDB ID [{{PDBlink}}4mai 4MAI] <cite>Hemsworth2013</cite>).''' On the left the overall structure is shown in cartoon representation with the surface of the protein shown in gray. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. On the right; at the top is a view of the active site showing the coordinatiuon of the copper ion by the “histidine brace”. Below are shown the active sites of ''Thermoascus aurantiacus'' [[AA9]] (PDB ID [{{PDBlink}}2yet 2YET] <cite>Quinlan2011</cite>) and ''Bacillus amyloliquefaciens'' [[AA10]] (PDB ID [{{PDBlink}}2yoy 2YOY] <cite>Hemsworth2013b</cite>) in the same orientation showing how the AA11 forms a hybrid active site between the two families.]]<br />
<br />
Though AA11 does not share significant sequence similarity to [[AA9]] and [[AA10]] proteins, the core fold of the protein is remarkably similar forming a typical ß-sandwich immunoglobulin like fold <cite>Hemsworth2013</cite>. The active site is at the centre of a slightly concave surface which, consistent with observations in [[AA10]]s <cite>Aachmann2012 Vaaje-Kolstad2012</cite>, has very few aromatic residues suggesting that it also primarily interacts with chitin via H-bonding interactions. Given that LPMOs are oxidoreductases there is increasing interest in the role of electron transport chains within these proteins <cite>Li2012</cite>. Internal tyrosine and tryptophan residues have been implicated in these roles in [[AA9]]s <cite>Li2012</cite> and [[AA10]]s <cite>Hemsworth2013c</cite> respectively. Similarly in AA11s it is possible to trace a path through the core of the protein using tryptophans, methionine and other hydrophilic residues to the distal face of the enzyme from the active site suggesting that there may be a similar electron transport path in AA11s though these are yet to be experimentally verified in LPMOs.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA11 from ''A. oryzae'' <cite>Hemsworth2013</cite>.<br />
;First demonstration of oxidative cleavage: AoAA11 was shown to oxidatively cleave squid pen chitin producing a mixture of aldonic acids, unmodified oligonucleotides and undistinguishable lactone/C4 oxidation products <cite>Hemsworth2013</cite>.<br />
;First 3-D structure: AA11 from ''A. oryzae'' with Zn2+ [{{PDBlink}}4mah 4MAH] & Cu(I) [{{PDBlink}}4mai 4MAI] <cite>Hemsworth2013</cite><br />
<br />
== References ==<br />
<biblio><br />
#Hemsworth2013 pmid=24362702<br />
#Horn2012 pmid=22747961<br />
#Kim2013 pmid=24344312<br />
#Hemsworth2013c pmid=23769965<br />
#Harris2010 pmid=20230050<br />
#Karkehabadi2008 pmid=18723026<br />
#Li2012 pmid=22578542<br />
#Quinlan2011 pmid=21876164<br />
#Hemsworth2013b pmid=23540833<br />
#Aachmann2012 pmid=23112164<br />
#Vaaje-Kolstad2012 pmid=22210154<br />
#Vaaje-Kolstad2005 pmid=15590674<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA11]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_11&diff=9854Auxiliary Activity Family 112014-01-20T16:48:47Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Gideon Davies^^^ & ^^^Glyn Hemsworth^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 11'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]] & [[AA10]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA11.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA11 family of Lytic Polysaccharide Mono-oxygenases (LPMOs) was identified using a bioinformatic 'module walking' approach <cite>Hemsworth2013</cite>. Many [[AA9]] proteins have C-terminal domains, which are often cellulose binding CBMs (eg. [[CBM1]]), but some have no known function (also see <cite>Horn2012</cite>). Sequence searches using one of these domains (termed X278) returned hits where the domain was fused with GH18 family members and another set of domains of unknown function. Closer examination of these domains revealed that they had a likely signal peptide followed by a histidine, typical of LPMOs, though they otherwise lacked significant sequence similarity to either [[AA9]] or [[AA10]] family members.<br />
<br />
The only AA11 to be isolated to to date is from ''Aspergillus oryzae'' which showed copper dependent oxidase activity on chitin in the presence of ascorbate. The observed products were a mixture of mainly aldonic acids and unmodified oligosaccharides with a small amount of a -2Da species, which may be C4 oxidation or the C1 lactone prior to ring opening. It is unknown at this stage whether AA11 family members will show any activity towards other substrates, whether they oxidise at positions other than C1 on the sugar ring or what the role of the X278 domain often found at the C-terminus is.<br />
<br />
== Kinetics and Mechanism ==<br />
AA11s, like [[AA9]]s and [[AA10]]s, are copper dependent mono-oxygenases but the chemical mechanism by which these enzymes perform the reaction is yet to be elucidated. Recent quantum mechanical simulations suggest that [[AA9]]s are likely to oxidise cellulose using a copper-oxyl, oxygen rebound mechanism <cite>Kim2013</cite> but further work is needed in this area. The differences in the copper coordination geometries between the families might further hint that they might use different mechanisms (Reviewed in <cite>Hemsworth2013c</cite>). As is the case for [[AA10]]s, the natural electron donor for AA11s is unknown, with no equivalent of cellobiose dehydrogenase yet discovered that is active on chitobiose. It is also unknown whether the N-terminal histidine in AA11s is N-methylated as is seen in [[AA9]]s and what effect this will have on the reaction performed by the enzyme. <br />
<br />
== Catalytic Residues ==<br />
Like [[AA9]] and [[AA10]], AA11 utilises copper, which it binds through the "histidine brace" consisting of the N-terminal histidine's amino and imidazole groups together with the imidazole sidechain of another histidine. It is unknown at this stage whether AA11s possess the methylated N-terminal histidine observed in [[AA9]]s as the protein was produced recombinantly in ''E. coli'' which does not carry out this modification. Like [[AA9]], a tyrosine sidechain is in close proximity to the copper ion but it does not directly coordinate the metal in this case. [[AA9]]s typically leave the opposite side to the tyrosine open to solvent where water molecules are observed coordinating the metal ion <cite>Harris2010 Karkehabadi2008 Li2012 Quinlan2011</cite>. In [[AA10]]s there is an alanine in this position, which causes a distortion in the copper coordination geometry away from octahedral <cite>Hemsworth2013b Vaaje-Kolstad2012 Vaaje-Kolstad2005 Aachmann2012</cite>. Interestingly AA11 has an alanine in a very similar position giving an active site structure somewhere between that of [[AA9]]s and [[AA10]]s. The structure was only determined with Cu(I) in the active site so the coordination geometry of Cu(II) was not directly observed but electron paramagnetic resonance spectroscopy confirmed that the copper coordination geometry lies somewhere between that of [[AA9]]s and [[AA10]]s <cite>Hemsworth2013</cite>.<br />
<br />
== Three-dimensional structures ==<br />
[[Image:AoAA11_Cazypedia.png|thumb|right|486x332px|'''Figure 1. Structure of AA11 from ''Aspergillus oryzae'' (PDB ID [{{PDBlink}}4mai 4MAI] <cite>Hemsworth2013</cite>).''' On the left the overall structure is shown in cartoon representation with the surface of the protein shown in gray. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. On the right; at the top is a view of the active site showing the coordinatiuon of the copper ion by the “histidine brace”. Below are shown the active sites of ''Thermoascus aurantiacus'' [[AA9]] (PDB ID [{{PDBlink}}2yet 2YET] <cite>Quinlan2011</cite>) and ''Bacillus amyloliquefaciens'' [[AA10]] (PDB ID [{{PDBlink}}2yoy 2YOY] <cite>Hemsworth2013b</cite>) in the same orientation showing how the AA11 forms a hybrid active site between the two families.]]<br />
<br />
Though AA11 does not share significant sequence similarity to [[AA9]] and [[AA10]] proteins, the core fold of the protein is remarkably similar forming a typical ß-sandwich immunoglobulin like fold <cite>Hemsworth2013</cite>. The active site is at the centre of a slightly concave surface which, consistent with observations in [[AA10]]s <cite>Aachmann2012 Vaaje-Kolstad2012</cite>, has very few aromatic residues suggesting that it also primarily interacts with chitin via H-bonding interactions. Given that LPMOs are oxidoreductases there is increasing interest in the role of electron transport chains within these proteins <cite>Li2012</cite>. Internal tyrosine and tryptophan residues have been implicated in these roles in [[AA9]]s <cite>Li2012</cite> and [[AA10]]s <cite>Hemsworth2013c</cite> respectively. Similarly in AA11s it is possible to trace a path through the core of the protein using tryptophans, methionine and other hydrophilic residues to the distal face of the enzyme from the active site suggesting that there may be a similar electron transport path in AA11s though these are yet to be experimentally verified in LPMOs.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA11 from ''A. oryzae'' <cite>Hemsworth2013</cite>.<br />
;First demonstration of oxidative cleavage: AoAA11 was shown to oxidatively cleave squid pen chitin producing a mixture of aldonic acids, unmodified oligonucleotides and undistinguishable lactone/C4 oxidation products <cite>Hemsworth2013</cite>.<br />
;First 3-D structure: AA11 from ''A. oryzae'' with Zn2+ [{{PDBlink}}4mah 4MAH] & Cu(I) [{{PDBlink}}4mai 4MAI] <cite>Hemsworth2013</cite><br />
<br />
== References ==<br />
<biblio><br />
#Hemsworth2013 pmid=24362702<br />
#Horn2012 pmid=22747961<br />
#Kim2013 pmid=24344312<br />
#Hemsworth2013c pmid=23769965<br />
#Harris2010 pmid=20230050<br />
#Karkehabadi2008 pmid=18723026<br />
#Li2012 pmid=22578542<br />
#Quinlan2011 pmid=21876164<br />
#Hemsworth2013b pmid=23540833<br />
#Aachmann2012 pmid=23112164<br />
#Vaaje-Kolstad2012 pmid=22210154<br />
#Vaaje-Kolstad2005 pmid=15590674<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA11]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_11&diff=9853Auxiliary Activity Family 112014-01-20T16:43:32Z<p>Glyn Hemsworth: </p>
<hr />
<div><br />
<!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Gideon Davies^^^ & ^^^Glyn Hemsworth^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 11'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]] & [[AA10]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA11.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA11 family of Lytic Polysaccharide Mono-oxygenases (LPMOs) was identified using a bioinformatic 'module walking' approach <cite>Hemsworth2013</cite>. Many [[AA9]] proteins have C-terminal domains, which are often cellulose binding CBMs (eg. [[CBM1]]), but some have no known function (also see <cite>Horn2012</cite>). Sequence searches using one of these domains (termed X278) returned hits where the domain was fused with GH18 family members and another set of domains of unknown function. Closer examination of these domains revealed that they had a likely signal peptide followed by a histidine, typical of LPMOs, though they otherwise lacked significant sequence similarity to either [[AA9]] or [[AA10]] family members.<br />
<br />
The only AA11 to be isolated to to date is from ''Aspergillus oryzae'' which showed copper dependent oxidase activity on chitin in the presence of ascorbate. The observed products were a mixture of mainly aldonic acids and unmodified oligosaccharides with a small amount of a -2Da species, which may be C4 oxidation or the C1 lactone prior to ring opening. It is unknown at this stage whether AA11 family members will show any activity towards other substrates, whether they oxidise at positions other than C1 on the sugar ring or what the role of the X278 domain often found at the C-terminus is.<br />
<br />
== Kinetics and Mechanism ==<br />
AA11s, like [[AA9]]s and [[AA10]]s, are copper dependent mono-oxygenases but the chemical mechanism by which these enzymes perform the reaction is yet to be elucidated. Recent quantum mechanical simulations suggest that [[AA9]]s are likely to oxidise cellulose using a copper-oxyl, oxygen rebound mechanism <cite>Kim2013</cite> but further work is needed in this area. The differences in the copper coordination geometries between the families might further hint that they might use different mechanisms (Reviewed in <cite>Hemsworth2013c</cite>). As is the case for [[AA10]]s, the natural electron donor for AA11s is unknown, with no equivalent of cellobiose dehydrogenase yet discovered that is active on chitobiose. It is also unknown whether the N-terminal histidine in AA11s is N-methylated as is seen in [[AA9]]s and what effect this will have on the reaction performed by the enzyme. <br />
<br />
== Catalytic Residues ==<br />
Like [[AA9]] and [[AA10]], AA11 utilises copper, which it binds through the "histidine brace" consisting of the N-terminal histidine's amino and imidazole groups together with the imidazole sidechain of another histidine. It is unknown at this stage whether AA11s possess the methylated N-terminal histidine observed in [[AA9]]s as the protein was produced recombinantly in ''E. coli'' which does not carry out this modification. Like [[AA9]], a tyrosine sidechain is in close proximity to the copper ion but it does not directly coordinate the metal in this case. [[AA9]]s typically leave the opposite side to the tyrosine open to solvent where water molecules are observed coordinating the metal ion <cite>Harris2010 Karkehabadi2008 Li2012 Quinlan2011</cite>. In [[AA10]]s there is an alanine in this position, which causes a distortion in the copper coordination geometry away from octahedral <cite>Hemsworth2013b</cite>. Interestingly AA11 has an alanine in a very similar position giving an active site structure somewhere between that of [[AA9]]s and [[AA10]]s. The structure was only determined with Cu(I) in the active site so the coordination geometry of Cu(II) was not directly observed but electron paramagnetic resonance spectroscopy confirmed that the copper coordination geometry lies somewhere between that of [[AA9]]s and [[AA10]]s <cite>Hemsworth2013</cite>.<br />
<br />
== Three-dimensional structures ==<br />
[[Image:AoAA11_Cazypedia.png|thumb|right|486x332px|'''Figure 1. Structure of AA11 from ''Aspergillus oryzae'' (PDB ID [{{PDBlink}}4mai 4MAI] <cite>Hemsworth2013</cite>).''' On the left the overall structure is shown in cartoon representation with the surface of the protein shown in gray. The active site is shown at the top of the structure with the copper ion shown as a sphere and surrounding active site residues shown as sticks with green carbon atoms. On the right; at the top is a view of the active site showing the coordinatiuon of the copper ion by the “histidine brace”. Below are shown the active sites of ''Thermoascus aurantiacus'' [[AA9]] (PDB ID [{{PDBlink}}2yet 2YET] <cite>Quinlan2011</cite>) and ''Bacillus amyloliquefaciens'' [[AA10]] (PDB ID [{{PDBlink}}2yoy 2YOY] <cite>Hemsworth2013b</cite>) in the same orientation showing how the AA11 forms a hybrid active site between the two families.]]<br />
<br />
Though AA11 does not share significant sequence similarity to [[AA9]] and [[AA10]] proteins, the core fold of the protein is remarkably similar forming a typical ß-sandwich immunoglobulin like fold <cite>Hemsworth2013</cite>. The active site is at the centre of a slightly concave surface which, consistent with observations in [[AA10]]s <cite>Aachmann2012 Vaaje-Kolstad2012</cite>, has very few aromatic residues suggesting that it also primarily interacts with chitin via H-bonding interactions. Given that LPMOs are oxidoreductases there is increasing interest in the role of electron transport chains within these proteins <cite>Li2012</cite>. Internal tyrosine and tryptophan residues have been implicated in these roles in [[AA9]]s <cite>Li2012</cite> and [[AA10]]s <cite>Hemsworth2013c</cite> respectively. Similarly in AA11s it is possible to trace a path through the core of the protein using tryptophans, methionine and other hydrophilic residues to the distal face of the enzyme from the active site suggesting that there may be a similar electron transport path in AA11s though these are yet to be experimentally verified in LPMOs.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA11 from ''A. oryzae'' <cite>Hemsworth2013</cite>.<br />
;First demonstration of oxidative cleavage: AoAA11 was shown to oxidatively cleave squid pen chitin producing a mixture of aldonic acids, unmodified oligonucleotides and undistinguishable lactone/C4 oxidation products <cite>Hemsworth2013</cite>.<br />
;First 3-D structure: AA11 from ''A. oryzae'' with Zn2+ [{{PDBlink}}4mah 4MAH] & Cu(I) [{{PDBlink}}4mai 4MAI] <cite>Hemsworth2013</cite><br />
<br />
== References ==<br />
<biblio><br />
#Hemsworth2013 pmid=24362702<br />
#Horn2012 pmid=22747961<br />
#Kim2013 pmid=24344312<br />
#Hemsworth2013c pmid=23769965<br />
#Harris2010 pmid=20230050<br />
#Karkehabadi2008 pmid=18723026<br />
#Li2012 pmid=22578542<br />
#Quinlan2011 pmid=21876164<br />
#Hemsworth2013b pmid=23540833<br />
#Aachmann2012 pmid=23112164<br />
#Vaaje-Kolstad2012 pmid=22210154<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA11]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=File:AoAA11_Cazypedia.png&diff=9852File:AoAA11 Cazypedia.png2014-01-20T16:32:30Z<p>Glyn Hemsworth: </p>
<hr />
<div></div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=Auxiliary_Activity_Family_11&diff=9850Auxiliary Activity Family 112014-01-20T14:51:28Z<p>Glyn Hemsworth: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Gideon Davies^^^ & ^^^Glyn Hemsworth^^^<br />
* [[Responsible Curator]]: ^^^Gideon Davies^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Auxiliary Activity Family 11'''<br />
|-<br />
|'''Clan''' <br />
|Structurally related to [[AA9]] & [[AA10]]<br />
|-<br />
|'''Mechanism'''<br />
|lytic oxidase<br />
|-<br />
|'''Active site residues'''<br />
|mononuclear copper ion<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}AA11.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The AA11 family of Lytic Polysaccharide Mono-oxygenases (LPMOs) was identified using a bioinformatic 'module walking' approach <cite>Hemsworth2013</cite>. Many [[AA9]] proteins have C-terminal domains, which are often cellulose binding CBMs (eg. [[CBM1]]), but some have no known function (also see <cite>Horn2012</cite>). Sequence searches using one of these domains (termed X278) returned hits where the domain was fused with GH18 family members and another set of domains of unknown function. Closer examination of these domains revealed that they had a likely signal peptide followed by a histidine, typical of LPMOs, though they otherwise lacked significant sequence similarity to either [[AA9]] or [[AA10]] family members.<br />
<br />
The only AA11 to be isolated to to date is from ''Aspergillus oryzae'' which showed copper dependent oxidase activity on chitin in the presence of ascorbate. The observed products were a mixture of mainly aldonic acids and unmodified oligosaccharides with a small amount of a -2Da species, which may be C4 oxidation or the C1 lactone prior to ring opening. It is unknown at this stage whether AA11 family members will show any activity towards other substrates, whether they oxidise at positions other than C1 on the sugar ring or what the role of the X278 domain often found at the C-terminus is.<br />
<br />
== Kinetics and Mechanism ==<br />
AA11s, like [[AA9]]s and [[AA10]]s, are copper dependent mono-oxygenases but the chemical mechanism by which these enzymes perform the reaction is yet to be elucidated. Recent quantum mechanical simulations suggest that [[AA9]]s are likely to oxidise cellulose using a copper-oxyl, oxygen rebound mechanism <cite>Kim2013</cite> but further work is needed in this area. The differences in the copper coordinatiion geometries between the families might further hint that they might use different mechanisms (Reviewed in <cite>Hemsworth2013c</cite>). As is the case for [[AA10]]s, the natural electron donor for AA11s is unknown, with no equivalent of cellobiose dehydrogenase yet discovered that is active on chitobiose. It is also unknown whether the N-terminal histidine in AA11s is N-methylated as is seen in [[AA9]]s and what effect this will have on the reaction performed by the enzyme. <br />
<br />
== Catalytic Residues ==<br />
Like [[AA9]] and [[AA10]], AA11 utilises copper, which it binds through the "histidine brace" consisting of the N-terminal histidine's amino and imidazole groups together with the imidazole sidechain of another histidine. It is unknown at this stage whether AA11s possess the methylated N-terminal histidine observed in [[AA9]]s as the protein was produced recombinantly in ''E. coli'' which does not carry out this modification. Like [[AA9]], a tyrosine sidechain is in close proximity to the copper ion but it does not directly coordinate the metal in this case. [[AA9]]s typically leave the opposite side to the tyrosine open to solvent where water molecules are observed coordinating the metal ion <cite>Harris2010 Karkehabadi2008 Li2012 Quinlan2011</cite>. In [[AA10]]s there is an alanine in this position, which causes a distortion in the copper coordination geometry away from octahedral <cite>Hemsworth2013b</cite>. Interestingly AA11 has an alanine in a very similar position giving an active site structure somewhere between that of [[AA9]]s and [[AA10]]s. The structure was only determined with Cu(I) in the active site so the coordination geometry of Cu(II) was not directly observed but electron paramagnetic resonance spectroscopy confirmed that the copper coordination geometry lies somewhere between that of [[AA9]]s and [[AA10]]s <cite>Hemsworth2013</cite>.<br />
<br />
== Three-dimensional structures ==<br />
Though AA11 does not share significant sequence similarity to [[AA9]] and [[AA10]] proteins, the core fold of the protein is remarkably similar forming a typical ß-sandwich immunoglobulin like fold <cite>Hemsworth2013</cite>. The active site is at the centre of a slightly concave surface which, consistent with observations in [[AA10]]s <cite>Aachmann2012 Vaaje-Kolstad2012</cite>, has very few aromatic residues suggesting that it also primarily interacts with chitin via H-bonding interactions. Given that LPMOs are oxidoreductases there is increasing interest in the role of electron transport chains within these proteins <cite>Li2012</cite>. Internal tyrosine and tryptophan residues have been implicated in these roles in [[AA9]]s <cite>Li2012</cite> and [[AA10]]s <cite>Hemsworth2013b</cite> respectively. Similarly in AA11s it is possible to trace a path through the core of the protein using tryptophans, methionine and other hydrophilic residues to the distal face of the enzyme from the active site suggesting that there may be a similar electron transport path in AA11s though these are yet to be experimentally verified in LPMOs.<br />
<br />
== Family Firsts ==<br />
;First family member identified: AA11 from ''A. oryzae'' <cite>Hemsworth2013</cite>.<br />
;First demonstration of oxidative cleavage: AoAA11 was shown to oxidatively cleave squid pen chitin producing a mixture of aldonic acids, unmodified oligonucleotides and undistinguishable lactone/C4 oxidation products <cite>Hemsworth2013</cite>.<br />
;First 3-D structure: AA11 from ''A. oryzae'' with Zn2+ [{{PDBlink}}4mah 4MAH] & Cu(I) [{{PDBlink}}4mai 4MAI] <cite>Hemsworth2013</cite><br />
<br />
== References ==<br />
<biblio><br />
#Hemsworth2013 pmid=24362702<br />
#Horn2012 pmid=22747961<br />
#Kim2013 pmid=24344312<br />
#Hemsworth2013c pmid=23769965<br />
#Harris2010 pmid=20230050<br />
#Karkehabadi2008 pmid=18723026<br />
#Li2012 pmid=22578542<br />
#Quinlan2011 pmid=21876164<br />
#Hemsworth2013b pmid=23769965<br />
#Aachmann2012 pmid=23112164<br />
#Vaaje-Kolstad2012 pmid=22210154<br />
<br />
</biblio><br />
<br />
[[Category:Auxiliary Activity Families|AA11]]</div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=User:Glyn_Hemsworth&diff=9825User:Glyn Hemsworth2014-01-09T12:19:28Z<p>Glyn Hemsworth: </p>
<hr />
<div><br />
[[File:GlynHemsworth.jpg|200px|right]]<br />
Glyn Hemsworth obtained his B.Sc. in Biochemistry from the University of Sheffield where he stayed on to complete his PhD under the supervision of Prof. Peter Artymiuk, primarily focussing on the structural characterisation of an unusual Flap Endonuclease homologue from ''Escherichia coli''. In 2009 he moved to the Structural Biology Laboratory at the University of York where he initially worked on dUTPases from ''Trypanosoma'' and ''Leishmania'' species with Prof Keith Wilson. In 2012 he moved into the carbohydrate field taking up his current post-doctoral position with Prof ^^^Gideon Davies^^^ where his major contributions have been in the structural and functional characterisation of lytic polysaccharide mono-oxygenases (LPMOs) <cite>Hemsworth2013</cite> in families [[AA10]] and the newly discovered [[AA11]]. He has determined the crystal structures of<br />
<br />
* [[AA10]] from ''Bacillus amyloliquefaciens'' <cite>Hemsworth2013a</cite><br />
* [[AA11]] from ''Aspergillus oryzae'' <cite>Hemsworth2013b</cite><br />
<br />
<br />
----<br />
<br />
<biblio><br />
#Hemsworth2013 pmid=23769965<br />
#Hemsworth2013a pmid=23540833<br />
#Hemsworth2013b pmid=24362702<br />
</biblio><br />
<br />
[[Category:Contributors|Hemsworth,Glyn]]<br />
<!-- ATTENTION: Make sure to replace "Lastname,Firstname" with your own name, for proper sorting of the Contributors page. --></div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=File:GlynHemsworth.jpg&diff=9824File:GlynHemsworth.jpg2014-01-09T12:18:10Z<p>Glyn Hemsworth: </p>
<hr />
<div></div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=User:Glyn_Hemsworth&diff=9823User:Glyn Hemsworth2014-01-09T12:13:19Z<p>Glyn Hemsworth: </p>
<hr />
<div><br />
Glyn Hemsworth obtained his B.Sc. in Biochemistry from the University of Sheffield where he stayed on to complete his PhD under the supervision of Prof. Peter Artymiuk, primarily focussing on the structural characterisation of an unusual Flap Endonuclease homologue from ''Escherichia coli''. In 2009 he moved to the Structural Biology Laboratory at the University of York where he initially worked on dUTPases from ''Trypanosoma'' and ''Leishmania'' species with Prof Keith Wilson. In 2012 he moved into the carbohydrate field taking up his current post-doctoral position with Prof ^^^Gideon Davies^^^ where his major contributions have been in the structural and functional characterisation of lytic polysaccharide mono-oxygenases (LPMOs) <cite>Hemsworth2013</cite> in families [[AA10]] and the newly discovered [[AA11]]. He has determined the crystal structures of<br />
<br />
* [[AA10]] from ''Bacillus amyloliquefaciens'' <cite>Hemsworth2013a</cite><br />
* [[AA11]] from ''Aspergillus oryzae'' <cite>Hemsworth2013b</cite><br />
<br />
<br />
----<br />
<br />
<biblio><br />
#Hemsworth2013 pmid=23769965<br />
#Hemsworth2013a pmid=23540833<br />
#Hemsworth2013b pmid=24362702<br />
</biblio><br />
<br />
[[Category:Contributors|Hemsworth,Glyn]]<br />
<!-- ATTENTION: Make sure to replace "Lastname,Firstname" with your own name, for proper sorting of the Contributors page. --></div>Glyn Hemsworthhttps://www.cazypedia.org/index.php?title=User:Glyn_Hemsworth&diff=9822User:Glyn Hemsworth2014-01-09T12:12:12Z<p>Glyn Hemsworth: Created page with " ---- Glyn Hemsworth obtained his B.Sc. in Biochemistry from the University of Sheffield where he stayed on to complete his PhD under the supervision of Prof. Peter Artymiuk,..."</p>
<hr />
<div><br />
----<br />
<br />
Glyn Hemsworth obtained his B.Sc. in Biochemistry from the University of Sheffield where he stayed on to complete his PhD under the supervision of Prof. Peter Artymiuk, primarily focussing on the structural characterisation of an unusual Flap Endonuclease homologue from ''Escherichia coli''. In 2009 he moved to the Structural Biology Laboratory at the University of York where he initially worked on dUTPases from ''Trypanosoma'' and ''Leishmania'' species with Prof Keith Wilson. In 2012 he moved into the carbohydrate field taking up his current post-doctoral position with Prof ^^^Gideon Davies^^^ where his major contributions have been in the structural and functional characterisation of lytic polysaccharide mono-oxygenases (LPMOs) <cite>Hemsworth2013</cite> in families [[AA10]] and the newly discovered [[AA11]]. He has determined the crystal structures of<br />
<br />
* [[AA10]] from ''Bacillus amyloliquefaciens'' <cite>Hemsworth2013a</cite><br />
* [[AA11]] from ''Aspergillus oryzae'' <cite>Hemsworth2013b</cite><br />
<br />
<br />
----<br />
<br />
<biblio><br />
#Hemsworth2013 pmid=23769965<br />
#Hemsworth2013a pmid=23540833<br />
#Hemsworth2013b pmid=24362702<br />
</biblio><br />
<br />
[[Category:Contributors|Hemsworth,Glyn]]<br />
<!-- ATTENTION: Make sure to replace "Lastname,Firstname" with your own name, for proper sorting of the Contributors page. --></div>Glyn Hemsworth