Auxiliary Activity Family 9 - Revision history

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

2021-12-18T21:18:46Z

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

Paul Harris at 18:07, 11 August 2014

2014-08-11T18:07:59Z

Harry Brumer: fixed broken image

2014-05-11T15:15:00Z

fixed broken image

Paul Harris at 05:18, 11 May 2014

2014-05-11T05:18:26Z

Harry Brumer: /* Substrate specificities */ Added AA11 link

2014-01-09T20:35:18Z

Substrate specificities: Added AA11 link

Harry Brumer: /* Substrate specificities */

2014-01-09T20:34:41Z

Substrate specificities

Paul Harris: /* References */

2014-01-09T20:13:05Z

References

Paul Harris: /* Kinetics and Mechanism */

2014-01-09T20:11:54Z

Kinetics and Mechanism

Paul Harris: /* Substrate specificities */

2014-01-09T20:01:45Z

Substrate specificities

Paul Harris: /* Kinetics and Mechanism */

2014-01-09T19:54:53Z

Kinetics and Mechanism

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 {{CuratorApproved}}
-* [[Author]]: ^^^Paul Harris^^^
+* [[Author]]: [[User:Paul Harris|Paul Harris]]
-* [[Responsible Curator]]:  ^^^Paul Harris^^^
+* [[Responsible Curator]]:  [[User:Paul Harris|Paul Harris]]
 ----

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 This family was originally placed among the glycoside hydrolases in family [[GH61]] based largely on a report of very weak endoglucanase activity of “Cel61A” from ''Trichoderma reesei'' using substrates such as carboxymethyl cellulose and ß-glucan <cite>Karlsson2001</cite>. Subsequently, other investigations seemed to confirm this result with other AA9 proteins (e.g.<cite>Bauer2006</cite>). It remains unclear whether the activities observed could be ascribed to AA9 or to low levels of contaminating proteins. It is now believed that AA9 proteins are not endoglucanases or glycoside hydrolases at all but rather lytic polysaccharide monooxygenases (LPMOs) that cleave polysaccharides by a novel oxidative mechanism (see below). Current published evidence indicates that cellulose is the preferred substrate, however many AA9 proteins show little or no activity with cellulose and may have other substrate specificities, or possibly very different co-factor requirements. This is a very large family and only a fraction of its members have been assayed for activity on cellulose and an even smaller number assayed on other abundant polysaccharide substrates such as chitin, starch and xylan. Given the structural and mechanistic similarities between AA9 and chitin-active [[AA10]] (formerly [[CBM33]]) and [[AA11]] proteins, and the ability of some [[AA10]] proteins to cleave cellulose as well as chitin, it would not be surprising if some members of the AA9 family had substrate specificities other than cellulose (see below). It is also not clear that polysaccharides would be the only potential substrates, and thus the term LPMO would prudently be reserved for those AA9 family members with proven lytic activity on polysaccharides.
-Current thinking posits that AA9 and [[AA10]] proteins act on relatively crystalline surface regions of their substrates and thereby create attachment sites and enhanced accessibility for the canonical glycoside hydrolases that subsequently further hydrolyze the substrate. This hypothesis has not yet been rigorously tested but is the most  parsimonious explanation for the ability of these proteins to enhance glycoside hydrolase action. The relatively flat active-site surface of these proteins is consistent with binding to an ordered polysaccharide surface, although the mechanism of binding appears to involve mostly H-bonding interactions in the case of [[AA10]] <cite>Aachmann2012 Vaaje-Kolstad2012</cite> versus stacking with planar aromatic residues for AA9 <cite>Li2012 Wu2013</cite>. Soluble polysaccharides have not been shown to be a substrate for any AA9 proteins except for one from Neurospora crassa <cite>Isaksen2013</cite>. This unusual AA9 was also recently shown to be active on a variety of non-cellulosic substrates including xyloglucans, ß-glucans and glucomannan, with xyloglucan possibly the "native" substrate. The primary substrate requirement is the presence of (1,4)-ß-D-glucan in the backbone. Oxidative attack apparently occurs at this linkage despite xylose or galactose substitutions <cite>Agger2014</cite>.
+Current thinking posits that AA9 and [[AA10]] proteins act on relatively crystalline surface regions of their substrates and thereby create attachment sites and enhanced accessibility for the canonical glycoside hydrolases that subsequently further hydrolyze the substrate. This hypothesis has not yet been rigorously tested but is the most  parsimonious explanation for the ability of these proteins to enhance glycoside hydrolase action. In support of this, the synergistic enhancement of cellulase activity by AA9 from ''Thermoascus aurantiacus'' correlates well with the accessible crystalline to amorphous cellulose ratio, and no synergy is seen with amorphous cellulose II or III <cite>Hu2014</cite>.The relatively flat active-site surface of these proteins is consistent with binding to an ordered polysaccharide surface, although the mechanism of binding appears to involve mostly H-bonding interactions in the case of [[AA10]] <cite>Aachmann2012 Vaaje-Kolstad2012</cite> versus stacking with planar aromatic residues for AA9 <cite>Li2012 Wu2013</cite>. Soluble polysaccharides have not been shown to be a substrate for any AA9 proteins except for one from Neurospora crassa <cite>Isaksen2013</cite>. This unusual AA9 was also recently shown to be active on a variety of non-cellulosic substrates including xyloglucans, ß-glucans and glucomannan, with xyloglucan possibly the "native" substrate. The primary substrate requirement is the presence of (1,4)-ß-D-glucan in the backbone. Oxidative attack apparently occurs at this linkage despite xylose or galactose substitutions <cite>Agger2014</cite>.
 == Kinetics and Mechanism ==
@@ Line 80: / Line 80: @@
 #Kim2014 pmid=24344312
 #Agger2014 pmid=24733907
 </biblio>
 [[Category:Auxiliary Activity Families|AA009]]

@@ Line 41: / Line 41: @@
 == Three-dimensional structures ==
-[[
+[[Image:Overall Structure.png|thumb|right|462x321px|'''Figure 1. Cartoon structure and transparent surface representation of "GH61A" from ''Thermoascus aurantiacus'' (PDB ID [{{PDBlink}}2yet 2YET] <cite>Quinlan2011</cite>).''' A bound copper atom at the active site is shown as a sphere on the top flat surface where substrate very likely binds. To the right is a view of the active site showing the axially elongated tetragonal coordination geometry of the Cu(II) ion. The "histidine brace" is comprised of the N-terminal amine and imidazole of the very highly conserved N-terminal methyl-histidine along with the imidazole of a second highhly conserved histidine. The fourth equatorial position is occupied by a water molecule H-bonded to a highly conserved glutamine that is important for function <cite>Harris2010</cite>. A somewhat distant phenolic oxygen of a very highly conserved tyrosine occupies one of the axial positions, and an additional water molecule occupies the other. In other AA9 structures the axial water position is either vacant or occupied by what appears to be a superoxide or peroxide and may thus be the site of oxygen binding <cite>Li2012</cite>.]]
 In the structures so far determined <cite>Harris2010 Karkehabadi2008 Li2012 Quinlan2011 Wu2013</cite>, the core of the catalytic domain adopts an immunoglobulin-like ß-sandwich fold with the individual ß-strands connected by long loops that have one to several helical insertions of variable length. Families AA9 and [[AA10]] share a common ß-sandwich fold and a similarly flat surface that contains the catalytic metal binding site. Different metal ions have been observed in different structures depending on which metal was most abundantly present in the crystallization buffer, however copper appears to be the "native" metal ion. In structures of AA9 published to date, the N-terminal histidine is methylated at N&epsilon;. The function of this methylation, if any, is unknown, but could serve to increase pKa of the histidine or to modulate redox activity. To date only the structures of the core catalytic domains have been solved. Many AA9 proteins have conserved C-terminal extensions beyond the obvious catalytic domain and these have no obvious identity to anything outside of the AA9 family (e.g. [http://www.uniprot.org/uniprot/N4XCE3 Uniprot N4XCE3]). Others have recognizable linker domains with a family 1 cellulose binding module (CBM1) or other conserved modules of unknown function.
@@ Line 81: / Line 79: @@
 #Isaksen2013 pmid=24324265
 #Kim2014 pmid=24344312
 #Agger2014 pmid=24733907
 </biblio>
 [[Category:Auxiliary Activity Families|AA009]]

@@ Line 28: / Line 28: @@
 == Substrate specificities ==
-This family was originally placed among the glycoside hydrolases in family [[GH61]] based largely on a report of very weak endoglucanase activity of “Cel61A” from ''Trichoderma reesei'' using substrates such as carboxymethyl cellulose and ß-glucan <cite>Karlsson2001</cite>. Subsequently, other investigations seemed to confirm this result with other AA9 proteins (e.g.<cite>Bauer2006</cite>). It remains unclear whether the activities observed could be ascribed to AA9 or to low levels of contaminating proteins. It is now believed that AA9 proteins are not endoglucanases or glycoside hydrolases at all but rather lytic polysaccharide monooxygenases (LPMOs) that cleave polysaccharides by a novel oxidative mechanism (see below). Current published evidence indicates that cellulose is the preferred substrate, however many AA9 proteins show little or no activity with cellulose and may have other substrate specificities, or possibly very different co-factor requirements. This is a very large family and only a fraction of its members have been assayed for activity on cellulose and an even smaller number assayed on other abundant polysaccharide substrates such as chitin, starch and xylan. Given the structural and mechanistic similarities between AA9 and chitin-active [[AA10]] (formerly [[CBM33]]) and [[AA11]] proteins, and the ability of some [[AA10]] proteins to cleave cellulose as well as chitin, it would not be surprising if some members of the AA9 family had substrate specificities other than cellulose. It is also not clear that polysaccharides would be the only potential substrates, and thus the term LPMO would prudently be reserved for those AA9 family members with proven lytic activity on polysaccharides.
+This family was originally placed among the glycoside hydrolases in family [[GH61]] based largely on a report of very weak endoglucanase activity of “Cel61A” from ''Trichoderma reesei'' using substrates such as carboxymethyl cellulose and ß-glucan <cite>Karlsson2001</cite>. Subsequently, other investigations seemed to confirm this result with other AA9 proteins (e.g.<cite>Bauer2006</cite>). It remains unclear whether the activities observed could be ascribed to AA9 or to low levels of contaminating proteins. It is now believed that AA9 proteins are not endoglucanases or glycoside hydrolases at all but rather lytic polysaccharide monooxygenases (LPMOs) that cleave polysaccharides by a novel oxidative mechanism (see below). Current published evidence indicates that cellulose is the preferred substrate, however many AA9 proteins show little or no activity with cellulose and may have other substrate specificities, or possibly very different co-factor requirements. This is a very large family and only a fraction of its members have been assayed for activity on cellulose and an even smaller number assayed on other abundant polysaccharide substrates such as chitin, starch and xylan. Given the structural and mechanistic similarities between AA9 and chitin-active [[AA10]] (formerly [[CBM33]]) and [[AA11]] proteins, and the ability of some [[AA10]] proteins to cleave cellulose as well as chitin, it would not be surprising if some members of the AA9 family had substrate specificities other than cellulose (see below). It is also not clear that polysaccharides would be the only potential substrates, and thus the term LPMO would prudently be reserved for those AA9 family members with proven lytic activity on polysaccharides.
-Current thinking posits that AA9 and [[AA10]] proteins act on relatively crystalline surface regions of their substrates and thereby create attachment sites and enhanced accessibility for the canonical glycoside hydrolases that subsequently further hydrolyze the substrate. This hypothesis has not yet been rigorously tested but is the most  parsimonious explanation for the ability of these proteins to enhance glycoside hydrolase action. The relatively flat active-site surface of these proteins is consistent with binding to an ordered polysaccharide surface, although the mechanism of binding appears to involve mostly H-bonding interactions in the case of [[AA10]] <cite>Aachmann2012 Vaaje-Kolstad2012</cite> versus stacking with planar aromatic residues for AA9 <cite>Li2012 Wu2013</cite>. Soluble polysaccharides have not been shown to be a substrate for any AA9 proteins except for one from Neurospora crassa <cite>Isaksen2013</cite>.
+Current thinking posits that AA9 and [[AA10]] proteins act on relatively crystalline surface regions of their substrates and thereby create attachment sites and enhanced accessibility for the canonical glycoside hydrolases that subsequently further hydrolyze the substrate. This hypothesis has not yet been rigorously tested but is the most  parsimonious explanation for the ability of these proteins to enhance glycoside hydrolase action. The relatively flat active-site surface of these proteins is consistent with binding to an ordered polysaccharide surface, although the mechanism of binding appears to involve mostly H-bonding interactions in the case of [[AA10]] <cite>Aachmann2012 Vaaje-Kolstad2012</cite> versus stacking with planar aromatic residues for AA9 <cite>Li2012 Wu2013</cite>. Soluble polysaccharides have not been shown to be a substrate for any AA9 proteins except for one from Neurospora crassa <cite>Isaksen2013</cite>. This unusual AA9 was also recently shown to be active on a variety of non-cellulosic substrates including xyloglucans, ß-glucans and glucomannan, with xyloglucan possibly the "native" substrate. The primary substrate requirement is the presence of (1,4)-ß-D-glucan in the backbone. Oxidative attack apparently occurs at this linkage despite xylose or galactose substitutions <cite>Agger2014</cite>.
 == Kinetics and Mechanism ==
@@ Line 41: / Line 41: @@
 == Three-dimensional structures ==
-[[Image:Overall Structure.png|thumb|right|462x321px|'''Figure 1. Cartoon structure and transparent surface representation of "GH61A" from ''Thermoascus aurantiacus'' (PDB ID [{{PDBlink}}2yet 2YET] <cite>Quinlan2011</cite>).''' A bound copper atom at the active site is shown as a sphere on the top flat surface where substrate very likely binds. To the right is a view of the active site showing the axially elongated tetragonal coordination geometry of the Cu(II) ion. The "histidine brace" is comprised of the N-terminal amine and imidazole of the very highly conserved N-terminal methyl-histidine along with the imidazole of a second highhly conserved histidine. The fourth equatorial position is occupied by a water molecule H-bonded to a highly conserved glutamine that is important for function <cite>Harris2010</cite>. A somewhat distant phenolic oxygen of a very highly conserved tyrosine occupies one of the axial positions, and an additional water molecule occupies the other. In other AA9 structures the axial water position is either vacant or occupied by what appears to be a superoxide or peroxide and may thus be the site of oxygen binding <cite>Li2012</cite>.]]
+[[
 In the structures so far determined <cite>Harris2010 Karkehabadi2008 Li2012 Quinlan2011 Wu2013</cite>, the core of the catalytic domain adopts an immunoglobulin-like ß-sandwich fold with the individual ß-strands connected by long loops that have one to several helical insertions of variable length. Families AA9 and [[AA10]] share a common ß-sandwich fold and a similarly flat surface that contains the catalytic metal binding site. Different metal ions have been observed in different structures depending on which metal was most abundantly present in the crystallization buffer, however copper appears to be the "native" metal ion. In structures of AA9 published to date, the N-terminal histidine is methylated at N&epsilon;. The function of this methylation, if any, is unknown, but could serve to increase pKa of the histidine or to modulate redox activity. To date only the structures of the core catalytic domains have been solved. Many AA9 proteins have conserved C-terminal extensions beyond the obvious catalytic domain and these have no obvious identity to anything outside of the AA9 family (e.g. [http://www.uniprot.org/uniprot/N4XCE3 Uniprot N4XCE3]). Others have recognizable linker domains with a family 1 cellulose binding module (CBM1) or other conserved modules of unknown function.
@@ Line 78: / Line 81: @@
 #Isaksen2013 pmid=24324265
 #Kim2014 pmid=24344312
 </biblio>
 [[Category:Auxiliary Activity Families|AA009]]

@@ Line 28: / Line 28: @@
 == Substrate specificities ==
-This family was originally placed among the glycoside hydrolases in family [[GH61]] based largely on a report of very weak endoglucanase activity of “Cel61A” from ''Trichoderma reesei'' using substrates such as carboxymethyl cellulose and ß-glucan <cite>Karlsson2001</cite>. Subsequently, other investigations seemed to confirm this result with other AA9 proteins (e.g.<cite>Bauer2006</cite>). It remains unclear whether the activities observed could be ascribed to AA9 or to low levels of contaminating proteins. It is now believed that AA9 proteins are not endoglucanases or glycoside hydrolases at all but rather lytic polysaccharide monooxygenases (LPMOs) that cleave polysaccharides by a novel oxidative mechanism (see below). Current published evidence indicates that cellulose is the preferred substrate, however many AA9 proteins show little or no activity with cellulose and may have other substrate specificities, or possibly very different co-factor requirements. This is a very large family and only a fraction of its members have been assayed for activity on cellulose and an even smaller number assayed on other abundant polysaccharide substrates such as chitin, starch and xylan. Given the structural and mechanistic similarities between AA9 and chitin-active [[AA10]] (formerly [[CBM33]]) and AA11 proteins, and the ability of some [[AA10]] proteins to cleave cellulose as well as chitin, it would not be surprising if some members of the AA9 family had substrate specificities other than cellulose. It is also not clear that polysaccharides would be the only potential substrates, and thus the term LPMO would prudently be reserved for those AA9 family members with proven lytic activity on polysaccharides.
+This family was originally placed among the glycoside hydrolases in family [[GH61]] based largely on a report of very weak endoglucanase activity of “Cel61A” from ''Trichoderma reesei'' using substrates such as carboxymethyl cellulose and ß-glucan <cite>Karlsson2001</cite>. Subsequently, other investigations seemed to confirm this result with other AA9 proteins (e.g.<cite>Bauer2006</cite>). It remains unclear whether the activities observed could be ascribed to AA9 or to low levels of contaminating proteins. It is now believed that AA9 proteins are not endoglucanases or glycoside hydrolases at all but rather lytic polysaccharide monooxygenases (LPMOs) that cleave polysaccharides by a novel oxidative mechanism (see below). Current published evidence indicates that cellulose is the preferred substrate, however many AA9 proteins show little or no activity with cellulose and may have other substrate specificities, or possibly very different co-factor requirements. This is a very large family and only a fraction of its members have been assayed for activity on cellulose and an even smaller number assayed on other abundant polysaccharide substrates such as chitin, starch and xylan. Given the structural and mechanistic similarities between AA9 and chitin-active [[AA10]] (formerly [[CBM33]]) and [[AA11]] proteins, and the ability of some [[AA10]] proteins to cleave cellulose as well as chitin, it would not be surprising if some members of the AA9 family had substrate specificities other than cellulose. It is also not clear that polysaccharides would be the only potential substrates, and thus the term LPMO would prudently be reserved for those AA9 family members with proven lytic activity on polysaccharides.
 Current thinking posits that AA9 and [[AA10]] proteins act on relatively crystalline surface regions of their substrates and thereby create attachment sites and enhanced accessibility for the canonical glycoside hydrolases that subsequently further hydrolyze the substrate. This hypothesis has not yet been rigorously tested but is the most  parsimonious explanation for the ability of these proteins to enhance glycoside hydrolase action. The relatively flat active-site surface of these proteins is consistent with binding to an ordered polysaccharide surface, although the mechanism of binding appears to involve mostly H-bonding interactions in the case of [[AA10]] <cite>Aachmann2012 Vaaje-Kolstad2012</cite> versus stacking with planar aromatic residues for AA9 <cite>Li2012 Wu2013</cite>. Soluble polysaccharides have not been shown to be a substrate for any AA9 proteins except for one from Neurospora crassa <cite>Isaksen2013</cite>.

@@ Line 35: / Line 35: @@
 It was known for many years from the patent literature (e.g. [{{PatentLink}}US20060005279 US20060005279], [{{PatentLink}}US20070077630 US20070077630], [{{PatentLink}}WO2005074647 WO2005074647]) and conference proceedings that some AA9 proteins could dramatically enhance the activity of canonical cellulase mixtures in the hydrolysis of lignocellulose, however this information was not published in a peer-reviewed journal until 2010 <cite>Harris2010</cite>. An explanation for this enhancement remained elusive for many years, but a first clue was the demonstration in the 2010 publication that metal ion is required for the enhancement and that the enhancement is not evident with relatively pure cellulose (Avicel) but only with pretreated lignocellulose such as acid-pretreated corn stover. In hindsight it became clear that the complex lignocellulosic substrate was providing an essential cofactor(s) that the Avicel did not. Subsequent studies, again first published in the patent literature ([{{PatentLink}}US20100159536 US20100159536]) and subsequently in journals <cite>Langston2011 Philips2011</cite>, showed that a combination of AA9 and cellobiose dehydrogenase could cleave pure cellulose. Additional work showed that a large number of small redox-active molecules such as gallate, ascorbate and catechol could replace cellobiose dehydrogenase in this regard, and both soluble and insoluble lignin and lignin derivatives might also act in this capacity <cite>Dimarogona2012</cite>. However, the “natural”  redox-active cofactors for AA9 proteins are currently unknown and may well vary depending on availability and the specific AA9 protein. Cellobiose dehydrogenase is often mentioned as a potential natural cofactor and indeed is often co-secreted with AA9 proteins. However there are many species of filamentous fungi (e.g. ''Paxillus involutus'', ''Talaromyces stipitatus'', ''Talaromyces thermophilus,'' and several ''Trichoderma'' (''Hypocrea'') species) that do not produce cellobiose dehydrogenase (based on lack of an obvious gene encoding it) and yet have multiple AA9-encoding genes. Nonetheless, for those fungi that do secrete cellobiose dehydrogenase, one of its main functions may be to synergize with AA9 proteins, presumably by acting as an electron donor.
-A key publication in 2010 <cite>Vaaje-Kolstad2010</cite> showed that [[AA10]], a structural relative of AA9, could oxidatively degrade crystalline chitin in the presence of metal ion and the reductant ascorbate, producing a mixture of oxidized and unoxidized oligosaccharides. Shortly thereafter, similar data were published on AA9 <cite>Quinlan2011 Westereng2011 Beeson2012</cite> (reviewed by <cite>HemsworthDavies2013 Dimarogona2013 Horn2012 LoLeggio2012</cite>) showing that these proteins are copper-dependent monooxygenases which are capable of inserting oxygen at the C-1 or C-4 (and perhaps C-6) position of the glycosidic bond in cellulose. The so-called type 1 LPMOs produce predominantly oxidation at C-1 (reducing end), probably initially producing an aldonolactone that spontaneously hydrolyzes to an aldonic acid. Type 2 LPMOs generate predominantly C-4 non-reducing end oxidized products (4-ketoaldolase and the hydrated gemdiol), and type 3 LPMOs appear to produce oxidized products at both the reducing and nonreducing ends <cite>Quinlan2011 Philips2011 Isaksen2013 Beeson2012</cite>. Results consistent with C-6 oxidation have also been reported <cite>Bey2013</cite>. Positioning of the substrate on the catalytic surface very likely determines the site of oxidation <cite>Li2012</cite>. The detailed interactions between AA9, bound metal ion, redox-active cofactor and substrate remain an area of active investigation, and additional surprises are likely. At present, the ability of AA9 proteins to oxidize cellulose is thought to arise from generation of oxidizing copper-oxygen species (superoxides or peroxides) at the active site by a mechanism that remains unclear (reviewed by Hemsworth et al. <cite>HemsworthDavies2013</cite>). Presumably Cu(I) at the active site activates a molecular oxygen and the activated oxygen species becomes incorporated into the cellulose chain. A reductant is then necessary to regenerate Cu(I) <cite>Beeson2012 Li2012</cite>.
+A key publication in 2010 <cite>Vaaje-Kolstad2010</cite> showed that [[AA10]], a structural relative of AA9, could oxidatively degrade crystalline chitin in the presence of metal ion and the reductant ascorbate, producing a mixture of oxidized and unoxidized oligosaccharides. Shortly thereafter, similar data were published on AA9 <cite>Quinlan2011 Westereng2011 Beeson2012</cite> (reviewed by <cite>HemsworthDavies2013 Dimarogona2013 Horn2012 LoLeggio2012</cite>) showing that these proteins are copper-dependent monooxygenases which are capable of inserting oxygen at the C-1 or C-4 (and perhaps C-6) position of the glycosidic bond in cellulose. The so-called type 1 LPMOs produce predominantly oxidation at C-1 (reducing end), probably initially producing an aldonolactone that spontaneously hydrolyzes to an aldonic acid. Type 2 LPMOs generate predominantly C-4 non-reducing end oxidized products (4-ketoaldolase and the hydrated gemdiol), and type 3 LPMOs appear to produce oxidized products at both the reducing and nonreducing ends <cite>Quinlan2011 Philips2011 Isaksen2013 Beeson2012</cite>. Results consistent with C-6 oxidation have also been reported <cite>Bey2013</cite>. Positioning of the substrate on the catalytic surface very likely determines the site of oxidation <cite>Li2012</cite>. The detailed interactions between AA9, bound metal ion, redox-active cofactor and substrate remain an area of active investigation, and additional surprises are likely. At present, the ability of AA9 proteins to oxidize cellulose is thought to arise from generation of oxidizing copper-oxygen species (superoxides or peroxides) at the active site by a mechanism that remains unclear (reviewed by Hemsworth et al. <cite>HemsworthDavies2013</cite>). Presumably Cu(I) at the active site activates a molecular oxygen and the activated oxygen species becomes incorporated into the cellulose chain. A reductant is then necessary to regenerate Cu(I) <cite>Beeson2012 Li2012</cite>. Quantum mechanical calculations favor a copper-oxyl radical mechanism for hydrogen abstraction and subsequent hydroxylation <cite>Kim2014</cite>.
 == Catalytic Residues ==