Auxiliary Activity Family 10 - Revision history

Harry Brumer: Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

2021-12-18T21:14:24Z

Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

Vincent Eijsink at 10:31, 20 January 2018

2018-01-20T10:31:23Z

Harry Brumer: adjusted figure formatting

2018-01-20T01:18:35Z

adjusted figure formatting

Harry Brumer: adjusted figure formatting

2018-01-20T01:15:43Z

adjusted figure formatting

Vincent Eijsink at 14:06, 19 January 2018

2018-01-19T14:06:00Z

Vincent Eijsink at 14:03, 19 January 2018

2018-01-19T14:03:39Z

Gustav Vaaje-Kolstad at 07:14, 19 January 2018

2018-01-19T07:14:03Z

Gustav Vaaje-Kolstad at 07:10, 19 January 2018

2018-01-19T07:10:29Z

Gustav Vaaje-Kolstad at 06:53, 19 January 2018

2018-01-19T06:53:41Z

Gustav Vaaje-Kolstad at 06:52, 19 January 2018

2018-01-19T06:52:32Z

@@ Line 1: / Line 1: @@
 <!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
 {{CuratorApproved}}
-* [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
+* [[Author]]: [[User:Vincent Eijsink|Vincent Eijsink]] and [[User:Gustav Vaaje-Kolstad|Gustav Vaaje-Kolstad]]
-* [[Responsible Curator]]:  ^^^Vincent Eijsink^^^
+* [[Responsible Curator]]:  [[User:Vincent Eijsink|Vincent Eijsink]]
 ----

@@ Line 1: / Line 1: @@
 <!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
-{{UnderConstruction}}
+{{CuratorApproved}}
 * [[Author]]: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
 * [[Responsible Curator]]:  ^^^Vincent Eijsink^^^

@@ Line 50: / Line 50: @@
 == Catalytic Residues and copper coordination ==
 [[Image:His_brace_4ALC_label.png|thumb|right|400px|'''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are shown as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low radiation dose X-ray crystallographic experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]
 Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was settled for AA9 LPMOs by Quinlan et al <cite>Quinlan2011</cite> and Phillips et al <cite>Phillips2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial", although this is not always formally correct (as exemplified by Fig. 3). One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.
-[[Image:AA10_diversityPNG.PNG|thumb|right|600px|'''''''''Figure 4. LPMO active sites.''' Active sites and their "second shell" of residues surrounding the histidine brace. CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite> ]]
+[[Image:AA10_diversityPNG.PNG|thumb|right|600px|'''Figure 4. LPMO active sites.''' Active sites and their "second shell" of residues surrounding the histidine brace. CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite>.]]
 The immediate environment of the copper binding site, sometimes referred to as the second shell, shows functionally conserved features that are known from experiment to be important for catalysis (e.g. <cite>Vaaje-Kolstad2005-2 Span2017 Forsberg2018</cite>). Further analysis and discussion of the roles of these residues awaits deeper insights into the catalytic mechanism of LPMOs and better assays for testing the true catalytic potential of LPMO variants. Figure 4 shows an example of such functionally conserved additional structural features and reference <cite>Forsberg2018</cite> describes a recent mutagenesis study on an AA10, including a discussion of the many pitfalls when trying to functionally interprete mutational effects.

@@ Line 41: / Line 41: @@
 == Kinetics and Mechanism ==
+[[Image:Rx_scheme_LPMOs.png|thumb|right|400px|'''Figure 2. Reaction schemes proposed for LPMOs in a) 2010 and b) 2017.''']]
 The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead to spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.
@@ Line 51: / Line 49: @@
 == Catalytic Residues and copper coordination ==
+[[Image:His_brace_4ALC_label.png|thumb|right|400px|'''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are shown as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low radiation dose X-ray crystallographic experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]
 Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was settled for AA9 LPMOs by Quinlan et al <cite>Quinlan2011</cite> and Phillips et al <cite>Phillips2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial", although this is not always formally correct (as exemplified by Fig. 3). One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.

@@ Line 65: / Line 65: @@
 ;First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various ''Streptomyces'' strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from ''Streptomyces olivaceoviridis'' a study published in 1994 <cite>Schnellman1994</cite>. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
 ;First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from ''S. marcescens'' is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three ''S. marsescens'' GH18 chitinases and a GH19 chitinase from ''Streptomyces coelicolor'' <cite>Vaaje-Kolstad2005-2</cite>. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
-;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
+;First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated <cite>Vaaje-Kolstad2010</cite>. This finding also represents the first demonstration of the true nature of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 <cite>Forsberg2011</cite>.
 ;First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium ''Serratia marcescens'', PDB ID [{{PDBlink}}2bem 2BEM].

@@ Line 54: / Line 54: @@
 [[Image:His_brace_4ALC_label.png|thumb|left|400px|''''''Figure 3. The active site of ''Ef''LPMO10 (''Ef''CBM33).''' The residues involved in, and in close proximity to, the histidine brace are shown in stick representation. The copper atom is shown as a golden sphere. Water molecules are shown as red spheres. The copper atom is coordinated in a trigonal bipyrimidal geometry. The figure is made from the structure of ''Ef''LPMO10 (also known as "''Ef''CBM33" from ''Enterococcus faecalis'' <cite>Gudmundsson2014</cite>) which was obtained by a low radiation dose X-ray crystallographic experiment that yielded an active site containing a copper atom in its Cu(II) state (normally copper atoms are reduced by X-ray photoreduction).]]
-Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite>Quinlan2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial", although this is not always formally correct (as exemplified by Fig. 3). One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.
+Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. 3) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was settled for AA9 LPMOs by Quinlan et al <cite>Quinlan2011</cite> and Phillips et al <cite>Phillips2011</cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial", although this is not always formally correct (as exemplified by Fig. 3). One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. 3) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this axial position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor an effect of its exact position, which varies among AA10 LPMOs.
 [[Image:AA10_diversityPNG.PNG|thumb|right|600px|'''''''''Figure 4. LPMO active sites.''' Active sites and their "second shell" of residues surrounding the histidine brace. CBP21 (dark grey) CelS2 (green), ScLPMO10B (orange) and TaLPMO9A (pink). The figure is adapted from <cite>Forsberg2014</cite> ]]

@@ Line 30: / Line 30: @@
 == Substrate specificities ==
 [[Image:CBP21_binding_chitin_modeled.jpg|thumb|right|400px|'''Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal.''' Please note that this complex has not been determined by direct experimentation nor computational molecular modelling.  The surfaces of residues known to interact with chitin <cite>Vaaje-Kolstad2005-1</cite> are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues.  This picture has been adapted from <cite>Horn2012</cite>.]]
-Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg 2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively.  AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.
+Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as [[Carbohydrate Binding Module Family 33]] members). They are known to cleave chitin <cite>Vaaje-Kolstad2010 Vaaje-Kolstad2012 Aachmann2012</cite> and cellulose <cite>Forsberg2011 Forsberg2014</cite>. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 <cite>Forsberg2014 Forsberg2018</cite>. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively.  AA10 modules often occur in combination with additional modules, in particular [[carbohydrate-binding modules]] (CBMs), but also catalytic domains such as GH18 chitinases <cite>Horn2012</cite>. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO <cite>Forsbergb2014 Crouch2016 Forsberg2018</cite>.
 Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of ''Serratia marcescens'' <cite>Fuchs1986, Suzuki1998</cite>, several ''Streptomyces'' species <cite>Zeltins1997 Kolbe1998 Saito2001</cite>, ''Bacillus amyloliquefaciens'' <cite>Chu2001</cite>,''Vibrio cholerae'' <cite>Wong2012</cite>, ''Pseudomonas aeruginosa'' <cite>Folders2000</cite> and ''Lacotococcus lactis'' <cite>Vaaje-Kolstad2009</cite>. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin <cite>Kolbe1998 Zeltins1997</cite>, beta-chitin <cite>Suzuki1998 Vaaje-Kolstad2005-1</cite>, both the alpha- and beta-chitin allomorphs <cite>Chu2001 Vaaje-Kolstad2009 Vaaje-Kolstad2012</cite> chitosan <cite>Saito2001</cite>, cellulose <cite>Walter2008 Forsberg2011</cite> and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc-containing glycoproteins or proteoglycans <cite>Sanches2011 Wong2012</cite>. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

@@ Line 42: / Line 42: @@
 == Kinetics and Mechanism ==
-[[Image:Rx_mech.png|thumb|right|600px|'''Figure 2. Reaction mechanisms proposed for LPMOs in a) 2010 and b) 2017.]]
+[[Image:Rx_scheme_LPMOs.png|thumb|right|600px|'''Figure 2. Reaction schemes proposed for LPMOs in a) 2010 and b) 2017.]]
 The catalytic mechanism of LPMOs is a matter of intense research and debate. Using labeled water and molecular oxygen, Vaaje-Kolstad et al <cite>Vaaje-Kolstad2010</cite> showed that one LPMO reaction requires two externally delivered electrons and molecular oxygen (Figure 2). Phillips ''et al'' <cite>Phillips2011</cite> pointed out that hydrogen abstraction (from the C1 or the C4) by a reactive oxygen species coordinated by the copper would be followed by hydroxylation and that this would lead to spontaneous cleavage of the glycosidic bond, leaving either the C1 or the C4 oxidized (yielding a lactone in equilibrium with carboxylic acid or a 4-keto group in equilibrium with a gemdiol, respectively <cite>Isaksen2014</cite>). The nature of the hydrogen abstracting oxygen species is not known. Current literature seems to be converging towards a Cu(II)-oxyl species <cite> Phillips2011 Kim2014 Bissaro2017 Bertini2018 Meier2018</cite>, but there are plausible alternatives that cannot be excluded, as outlined in e.g. <cite>Beeson2015 Walton2016 Bissaro2017 Meier2018</cite>.