Glycoside Hydrolase Family 12 - Revision history

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

2021-12-18T21:18:23Z

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

Spencer Williams at 11:57, 6 September 2015

2015-09-06T11:57:37Z

Spencer Williams at 11:57, 6 September 2015

2015-09-06T11:57:13Z

Spencer Williams at 11:56, 6 September 2015

2015-09-06T11:56:37Z

Spencer Williams at 23:05, 2 August 2015

2015-08-02T23:05:32Z

Harry Brumer: added Gilkes1991 ref. for original Cellulase Family H name.

2015-07-29T15:52:22Z

added Gilkes1991 ref. for original Cellulase Family H name.

Harry Brumer: /* Catalytic Residues */

2015-07-29T15:35:04Z

Catalytic Residues

Harry Brumer: /* Catalytic Residues */

2015-07-29T15:34:50Z

Catalytic Residues

Harry Brumer at 15:32, 29 July 2015

2015-07-29T15:32:49Z

Harry Brumer: changed author name order

2015-07-28T18:16:55Z

changed author name order

@@ Line 1: / Line 1: @@
 {{CuratorApproved}}
-* [[Author]]s: ^^^Gerlind Sulzenbacher^^^ and ^^^Mats Sandgren^^^
+* [[Author]]s: [[User:Gerlind Sulzenbacher|Gerlind Sulzenbacher]] and [[User:Mats Sandgren|Mats Sandgren]]
-* [[Responsible Curator]]:  ^^^Gerlind Sulzenbacher^^^
+* [[Responsible Curator]]:  [[User:Gerlind Sulzenbacher|Gerlind Sulzenbacher]]
 ----

@@ Line 60: / Line 60: @@
 ;First [[catalytic nucleophile]] identification: The catalytic nucleophile was first identified in ''Streptomyces lividans'' CelB2 by trapping of a glycosyl-enzyme intermediate and X-ray structure determination <cite>Sulzenbacher1999</cite> and peptide mapping using LC-MS/MS <cite>Zechel1998</cite>.
-;First [[general acid/base]] residue identification: inferred from homology with family [[GH11]] enzymes.
+;First [[general acid/base]] residue identification: Inferred from homology with family [[GH11]] enzymes.
 ;First 3-D structure: The crystal structure of ''Streptomyces lividans'' CelB2 <cite>Sulzenbacher1997</cite>.

@@ Line 56: / Line 56: @@
 == Family Firsts ==
-;First stereochemistry determination: ''Humicola insolens'' endoglucanase 3 by <sup>1<</sup>H NMR  <cite>Schou1993</cite>.
+;First stereochemistry determination: ''Humicola insolens'' endoglucanase 3 by <sup>1</sup>H NMR  <cite>Schou1993</cite>.
 ;First [[catalytic nucleophile]] identification: The catalytic nucleophile was first identified in ''Streptomyces lividans'' CelB2 by trapping of a glycosyl-enzyme intermediate and X-ray structure determination <cite>Sulzenbacher1999</cite> and peptide mapping using LC-MS/MS <cite>Zechel1998</cite>.

@@ Line 31: / Line 31: @@
 == Kinetics and Mechanism ==
-GH12 enzymes are [[retaining]] enzymes, as first shown by NMR studies <cite>Schou1993</cite> on endoglucanase 3 from ''Humicola insolens'', and is believed to follow a classical [[Koshland double-displacement mechanism]] in which a glycosyl-enzyme intermediate is formed and subsequently this intermediate is hydrolysed via an oxocarbenium-ion [[transition state]]. No detailed studies involving both steady state and pre-steady state kinetic have yet been reported for GH12.
+GH12 enzymes are [[retaining]] enzymes, as first shown by NMR studies <cite>Schou1993</cite> on endoglucanase 3 from ''Humicola insolens''. They are believed to follow a classical [[Koshland double-displacement mechanism]] in which a glycosyl-enzyme intermediate is formed and subsequently this intermediate is hydrolysed via an oxocarbenium-ion [[transition state]].
 == Catalytic Residues ==
-The [[catalytic nucleophile]] and the [[general acid/base]] residues of GH12 members (previously known as "family H cellulases" <cite>Gilkes1991 Torronen1993</cite>) was initially predicted by sequence homology to the xylanase members of [[GH11]] <cite>Torronen1993</cite>, a glycoside hydrolase family where the [[catalytic nucleophile]] was first identified in the ''Bacillus circulans'' [[endo]]-xylanase through trapping of the 2-deoxy-2-fluoroxylobiosyl-enzyme [[intermediate]] and subsequent peptide mapping using LC-MS/MS <cite>Miao1994</cite>. [[GH11]] and GH12 together form [[clan]] GH-C. The prediction of the [[catalytic nucleophile]] and the [[general acid/base]] residues of family GH12 enzymes was later confirmed when the first three dimensional structure of a family GH12 enzyme was determined, that of ''Streptomyces lividans'' CelB2 <cite>Sulzenbacher1997</cite>. Subsequently, the [[catalytic nucleophile]] in ''Streptomyces lividans'' CelB2 was assigned as Glu 120 by using the same labeling strategy used for detecting the [[catalytic nucleophile]] of family [[GH11]] <cite>Zechel1998</cite>.
+Families [[GH11]] and GH12 together form [[clan]] GH-C. The [[catalytic nucleophile]] and the [[general acid/base]] residues of family GH12 members (previously known as "family H cellulases" <cite>Gilkes1991 Torronen1993</cite>) was initially predicted by sequence homology to the xylanase members of [[GH11]] <cite>Torronen1993</cite>. Within family [[GH11]] the [[catalytic nucleophile]] was first identified in the ''Bacillus circulans'' [[endo]]-xylanase through trapping of the 2-deoxy-2-fluoroxylobiosyl-enzyme [[intermediate]] and subsequent peptide mapping using LC-MS/MS <cite>Miao1994</cite>. The prediction of the [[catalytic nucleophile]] and the [[general acid/base]] residues of family GH12 enzymes was later confirmed when the first three dimensional structure of a family GH12 enzyme was determined, that of ''Streptomyces lividans'' CelB2 <cite>Sulzenbacher1997</cite>. Subsequently, the [[catalytic nucleophile]] in ''Streptomyces lividans'' CelB2 was assigned as Glu 120 by using the same labeling strategy used for identifying the [[catalytic nucleophile]] of family [[GH11]] <cite>Zechel1998</cite>.
 == Three-dimensional structures ==
-[[File:gh12_1.png|thumb|300px|right|'''Figure 1: ''Streptomyces lividans'' CelB2 in complex with a 2-deoxy-2-fluorocellotrioside (PDB [{{PDBlink}}2nrl 2NLR]). ''' Two distinct species were observed in the structure: the glycosyl-enzyme intermediate with the mechanism-based inhibitor covalently linked to the nucleophile Glu 120, and the hydrolysis product, 2-deoxy-2-fluorocellotriose. ''Colour coding:'' CelB2 in rainbow colour-ramping from the N-terminus in blue to the C-terminus in red; catalytic residues in magenta; bound oligosaccharide in black]]
+[[File:gh12_1.png|thumb|300px|right|'''Figure 1: ''Streptomyces lividans'' CelB2 in complex with a 2-deoxy-2-fluorocellotrioside (PDB [{{PDBlink}}2nrl 2NLR]). ''' Two distinct species were modelled in the structure: the glycosyl-enzyme intermediate with the mechanism-based inhibitor covalently linked to the nucleophile Glu120, and the hydrolysis product, 2-deoxy-2-fluorocellotriose. ''Colour coding:'' CelB2 in rainbow colour-ramping from the N-terminus in blue to the C-terminus in red; catalytic residues in magenta; bound oligosaccharide in black]]
 Enzymes from family GH12 adopt a “jelly-roll” fold with two twisted, mostly antiparallel &beta;-sheets stacking on top of each other and enclosing a long substrate-binding groove located on the concave face of the sheet (Fig. 1). A single &alpha;-helix packs against the convex surface of the outer &beta;-sheet. GH12 members of bacterial origin feature an additional two-stranded &beta;-sheet, stabilized by a disulfide bridge, located at the rim of the substrate binding groove and providing an additional substrate binding platform as compared to the eukaryotic counterparts.
-The first structures for family GH12 were that of ''Streptomyces lividans'' CelB2 in the apo-form <cite>Sulzenbacher1997</cite> and in the form of a glycosyl-enzyme intermediate <cite>Sulzenbacher1999</cite>. These structures confirmed previous predictions, based on hydrophobic cluster analysis <cite>Torronen1993</cite>, that GH12 enzymes would display significant structural similarity with family [[GH11]] xylanases and gave rise to the creation of [[clan]] GH-C, which so far comprises only the two above families. The first structure of a fungal GH12 enzyme was provided shortly after, for ''Trichoderma reesei'' (formerly ''Hypocrea jecorina'') Cel12A <cite>Sandgren2001</cite>. To date structural models, both in the apo-form and in complex with oligosaccharides, are available for sixteen family members.
+The first structures for family GH12 were that of ''Streptomyces lividans'' CelB2 in the apo-form <cite>Sulzenbacher1997</cite> and in the form of a glycosyl-enzyme intermediate <cite>Sulzenbacher1999</cite>. These structures confirmed previous predictions, based on hydrophobic cluster analysis <cite>Torronen1993</cite>, that GH12 enzymes would display significant structural similarity with family [[GH11]] xylanases and gave rise to the creation of [[clan]] GH-C, which so far comprises only these two families. The first structure of a fungal GH12 enzyme was determined shortly thereafter, for ''Trichoderma reesei'' (formerly ''Hypocrea jecorina'') Cel12A <cite>Sandgren2001</cite>. To date structural models, both in the apo-form and in complex with oligosaccharides, are available for sixteen family members.
-From the various complex structures it can be seen that the substrate-binding grooves of family GH12 enzymes harbour six binding sites, from -4 to +2, lined by numerous aromatic residues providing binding platforms for the individual pyranose units. This is consistent with the endoglucanase activity of these enzymes, exhibiting low catalytic efficiency towards short chain substrates <cite>Karlsson2002</cite>. The [[catalytic nucleophile]] Glu120 (''S. lividans'' CelB2 numbering) and the [[general acid/base]] Glu203 are in close proximity of an invariant aspartate, which might have an important role in catalysis. This catalytic triad is reminiscent of the catalytic centre of family GH7 enzymes. GH12 family members, like those of family [[GH7]], have the acid/base residue situated 'syn' to the pyranoside 0-5-C-1 bond, and are thus classified as syn-protonators.
+The substrate-binding grooves of family GH12 enzymes harbour six binding sites, from -4 to +2, lined by numerous aromatic residues providing binding platforms for the individual pyranose units. This is consistent with the endoglucanase activity of these enzymes, exhibiting low catalytic efficiency towards short chain substrates <cite>Karlsson2002</cite>. The [[catalytic nucleophile]] Glu120 (''S. lividans'' CelB2 numbering) and the [[general acid/base]] Glu203 are in close proximity of an invariant aspartate, which might have an important role in catalysis. This catalytic triad is reminiscent of the catalytic centre of family [[GH7]] enzymes. GH12 family members, like those of family [[GH7]], have the acid/base residue situated 'syn' to the pyranoside 0-5-C-1 bond, and are thus classified as [[Syn/anti_lateral_protonation|syn-protonators]].
 [[File:gh12_2.png|thumb|300px|right|'''Figure 2: Xyloglucanase XG12 from ''Bacillus licheniformis'' in complex with xyloglucan (PDB [{{PDBlink}}}2jen 2JEN]).'''  &alpha;-1,6-xylose substitutions are present at subsites -3, -2, +1 and +2.]]
-For xyloglucan specific enzymes of the family, structural and functional studies of xyloglucanase XG12 from ''Bacillus licheniformis'' <cite>Gloster2007</cite> and the xyloglucan specific endo-&beta;-1,4-glucanase XEG1 from ''Aspergillus aculeatus KSM 50'' <cite>Yoshizawa2012</cite> have shown that &alpha;-1,6-xylose substitutions can be tolerated at subsites -3, -2, +1 and +2 (Fig. 2). Certain GH12 enzymes exhibit &beta;-1,3-1,4-glucanase activity and the crystal structure of ''Humicola grisea'' Cel12A in complex with a mixed linkage oligosaccharide arising from a transglycosylation reaction revealed that the &beta;-1,3-linkage can be accommodated with ease between subsites -3 and -2 <cite>Sandgren2004</cite>. It has been speculated that a long loop crossing the substrate-binding groove at its reducing end, known as the “cord” and conserved in all [[clan]] GH-C members, might undergo conformational changes upon substrate binding <cite>Torronen1994</cite>, but the available GH12 complex structures support the view that the role of this loop is to deliver residues for substrate binding.
+Structural and function studies of several xyloglucan-specific enzymes of the family, including xyloglucanase XG12 from ''Bacillus licheniformis'' <cite>Gloster2007</cite> and the xyloglucan specific endo-&beta;-1,4-glucanase XEG1 from ''Aspergillus aculeatus KSM 50'' <cite>Yoshizawa2012</cite>, have shown that &alpha;-1,6-xylose substitutions can be tolerated at subsites -3, -2, +1 and +2 (Fig. 2). Certain GH12 enzymes exhibit &beta;-1,3-1,4-glucanase activity and the crystal structure of ''Humicola grisea'' Cel12A in complex with a mixed linkage oligosaccharide arising from a transglycosylation reaction revealed that the &beta;-1,3-linkage can be accommodated between subsites -3 and -2 <cite>Sandgren2004</cite>. It has been speculated that a long loop crossing the substrate-binding groove at its reducing end, known as the “cord” and conserved in all [[clan]] GH-C members, might undergo conformational changes upon substrate binding <cite>Torronen1994</cite>, but the available GH12 complex structures support the view that the role of this loop is to deliver residues for substrate binding.
 The crystal structure of ''Aspergillus aculeatus'' XEG1 in complex with the glycoside hydrolase inhibitory protein (GHIP) from carrot, EDGP, reveals a mode of inhibition distinct from that observed previously for the inhibition of a family [[GH11]] xylanase by a wheat protein <cite>Payan2004</cite>. In the XEG1-GHIP complex the inhibitor mimics a xyloglucan substrate and inserts two conserved arginine residues into the active site, which establish salt bridges with the carboxylate groups of the catalytic centre (Fig. 3) <cite>Yoshizawa2012</cite>).
@@ Line 56: / Line 56: @@
 == Family Firsts ==
-;First sterochemistry determination: ''Humicola insolens'' endoglucanase 3 by <sup>1<</sup>NMR  <cite>Schou1993</cite>.
+;First stereochemistry determination: ''Humicola insolens'' endoglucanase 3 by <sup>1<</sup>H NMR  <cite>Schou1993</cite>.
 ;First [[catalytic nucleophile]] identification: The catalytic nucleophile was first identified in ''Streptomyces lividans'' CelB2 by trapping of a glycosyl-enzyme intermediate and X-ray structure determination <cite>Sulzenbacher1999</cite> and peptide mapping using LC-MS/MS <cite>Zechel1998</cite>.

@@ Line 28: / Line 28: @@
 == Substrate specificities ==
-The substrate specificities found among the [[glycoside hydrolases]] of family 12 are: ''[[endo]]''-&beta;-1,4-glucanase (EC [{{EClink}}3.2.1.4 3.2.1.4]), xyloglucan ''[[endo]]''-hydrolase (EC [{{EClink}}3.2.1.151 3.2.1.151]), ''[[endo]]''-&beta;-1,3-1,4-glucanase (EC [{{EClink}}3.2.1.73 3.2.1.73]).  Xyloglucan ''[[endo]]''-transglycosylase (XET, EC [{{EClink}}2.4.1.207 2.4.1.207]) activity has been observed in a single fungal GH12 member (GenBank [http://www.ncbi.nlm.nih.gov/protein/AAN89225.1 AAN89225.1]) using a XET-specific screen <cite>Nielsen2002</cite>, although this may represent a side activity of a predominant xyloglucan ''[[endo]]''-hydrolase <cite>Gilbert2008 Eklof2010</cite>.
+The substrate specificities found among the [[glycoside hydrolases]] of family 12 include: ''[[endo]]''-&beta;-1,4-glucanase (EC [{{EClink}}3.2.1.4 3.2.1.4]), xyloglucan ''[[endo]]''-hydrolase (EC [{{EClink}}3.2.1.151 3.2.1.151]), and ''[[endo]]''-&beta;-1,3-1,4-glucanase (EC [{{EClink}}3.2.1.73 3.2.1.73]).  Xyloglucan ''[[endo]]''-transglycosylase (XET, EC [{{EClink}}2.4.1.207 2.4.1.207]) activity has been observed in a single fungal GH12 member (GenBank [http://www.ncbi.nlm.nih.gov/protein/AAN89225.1 AAN89225.1]) using a XET-specific screen <cite>Nielsen2002</cite>, although this may represent a side activity of a predominant xyloglucan ''[[endo]]''-hydrolase <cite>Gilbert2008 Eklof2010</cite>.
 == Kinetics and Mechanism ==
-GH12 enzymes are [[retaining]] enzymes, as first shown by NMR studies <cite>Schou1993</cite> on endoglucanase 3 from ''Humicola insolens'', and is believed to follow a classical [[Koshland double-displacement mechanism]] in which a glycosyl-enzyme intermediate is formed and subsequently this intermediate is hydrolysed via oxocarbenium-ion [[transition state]]s. No detailed studies involving both steady state and pre-steady state kinetic have yet been reported for GH12.
+GH12 enzymes are [[retaining]] enzymes, as first shown by NMR studies <cite>Schou1993</cite> on endoglucanase 3 from ''Humicola insolens'', and is believed to follow a classical [[Koshland double-displacement mechanism]] in which a glycosyl-enzyme intermediate is formed and subsequently this intermediate is hydrolysed via an oxocarbenium-ion [[transition state]]. No detailed studies involving both steady state and pre-steady state kinetic have yet been reported for GH12.
 == Catalytic Residues ==
-The [[catalytic nucleophile]] and the [[general acid/base]] catalyst of GH12 members (previously known as "family H cellulases" <cite>Gilkes1991 Torronen1993</cite>) was initialy predicted by sequence homology to the xylanase members of [[GH11]] <cite>Torronen1993</cite>, a glycoside hydrolase family where the [[catalytic nucleophile]] was first identified in the ''Bacillus circulans'' [[endo]]-xylanase through trapping of the 2-deoxy-2-fluoroxylobiosyl-enzyme [[intermediate]] and subsequent peptide mapping via LC-MS/MS technologies <cite>Miao1994</cite>. [[GH11]] and GH12 together form [[clan]] GH-C. The prediction of the [[catalytic nucleophile]] and the [[general acid/base]] of GH family 12 enzymes was later confirmed to be correct when the first three dimensional structure of a GH family 12 enzyme was determined, that of ''Streptomyces lividans'' CelB2 <cite>Sulzenbacher1997</cite>. The [[catalytic nucleophile]] was subsequentialy confirmed in ''Streptomyces lividans'' CelB2 to be Glu 120 by using the same labeling strategy used for detecting the [[catalytic nucleophile]] of family [[GH11]] <cite>Zechel1998</cite>.
+The [[catalytic nucleophile]] and the [[general acid/base]] residues of GH12 members (previously known as "family H cellulases" <cite>Gilkes1991 Torronen1993</cite>) was initially predicted by sequence homology to the xylanase members of [[GH11]] <cite>Torronen1993</cite>, a glycoside hydrolase family where the [[catalytic nucleophile]] was first identified in the ''Bacillus circulans'' [[endo]]-xylanase through trapping of the 2-deoxy-2-fluoroxylobiosyl-enzyme [[intermediate]] and subsequent peptide mapping using LC-MS/MS <cite>Miao1994</cite>. [[GH11]] and GH12 together form [[clan]] GH-C. The prediction of the [[catalytic nucleophile]] and the [[general acid/base]] residues of family GH12 enzymes was later confirmed when the first three dimensional structure of a family GH12 enzyme was determined, that of ''Streptomyces lividans'' CelB2 <cite>Sulzenbacher1997</cite>. Subsequently, the [[catalytic nucleophile]] in ''Streptomyces lividans'' CelB2 was assigned as Glu 120 by using the same labeling strategy used for detecting the [[catalytic nucleophile]] of family [[GH11]] <cite>Zechel1998</cite>.
 == Three-dimensional structures ==
-[[File:gh12_1.png|thumb|300px|right|'''Figure 1: ''Streptomyces lividans'' CelB2 in complex with a 2-deoxy-2-fluorocellotrioside (PDB [{{PDBlink}}2nrl 2NLR]).''' Two distinct species were observed in the crystal: the glycosyl-enzyme intermediate with the mechanism-based inhibitor covalently linked to the nucleophile Glu 120, and the reaction product 2-deoxy-2-fluoro-cellotriose. ''Colour coding:'' CelB2 in rainbow colour-ramping from the N-terminus in blue to the C-terminus in red; catalytic residues in magenta; bound oligosaccharide in black]]
+[[File:gh12_1.png|thumb|300px|right|'''Figure 1: ''Streptomyces lividans'' CelB2 in complex with a 2-deoxy-2-fluorocellotrioside (PDB [{{PDBlink}}2nrl 2NLR]). ''' Two distinct species were observed in the structure: the glycosyl-enzyme intermediate with the mechanism-based inhibitor covalently linked to the nucleophile Glu 120, and the hydrolysis product, 2-deoxy-2-fluorocellotriose. ''Colour coding:'' CelB2 in rainbow colour-ramping from the N-terminus in blue to the C-terminus in red; catalytic residues in magenta; bound oligosaccharide in black]]
-Enzymes from family GH12 adopt a “jelly-roll” fold with two twisted, mostly antiparallel &beta;-sheets stacking on top of each other and enclosing a long substrate-binding groove located on the concave face of the sheet (Fig. 1). A single &alpha;-helix packs against the convex surface of the outer &beta;-sheet. GH12 members of bacterial origin feature an additional two-stranded &beta;-sheet, stabilized by a disulphide bridge, located at the rim of the substrate binding groove and providing an additional substrate binding platform as compared to the eukaryotic counterparts.
+Enzymes from family GH12 adopt a “jelly-roll” fold with two twisted, mostly antiparallel &beta;-sheets stacking on top of each other and enclosing a long substrate-binding groove located on the concave face of the sheet (Fig. 1). A single &alpha;-helix packs against the convex surface of the outer &beta;-sheet. GH12 members of bacterial origin feature an additional two-stranded &beta;-sheet, stabilized by a disulfide bridge, located at the rim of the substrate binding groove and providing an additional substrate binding platform as compared to the eukaryotic counterparts.
-The first structural template provided for family GH12 was that of ''Streptomyces lividans'' CelB2 in the apo-form <cite>Sulzenbacher1997</cite> and in the form of a glycosyl-enzyme intermediate <cite>Sulzenbacher1999</cite>. This first structure confirmed previous predictions, based on hydrophobic cluster analysis <cite>Torronen1993</cite>, that GH12 enzymes would display significant structural similarity with family [[GH11]] xylanases and gave rise to the creation of [[clan]] GH-C, which so far comprises only the two above families. The first structure of a fungal GH12 enzyme was provided shortly after for ''Trichoderma reesei'' (formerly ''Hypocrea jecorina'') Cel12A <cite>Sandgren2001</cite>. To date structural models, both in the apo-form and in complex with oligosaccharides, are available for sixteen family members.
+The first structures for family GH12 were that of ''Streptomyces lividans'' CelB2 in the apo-form <cite>Sulzenbacher1997</cite> and in the form of a glycosyl-enzyme intermediate <cite>Sulzenbacher1999</cite>. These structures confirmed previous predictions, based on hydrophobic cluster analysis <cite>Torronen1993</cite>, that GH12 enzymes would display significant structural similarity with family [[GH11]] xylanases and gave rise to the creation of [[clan]] GH-C, which so far comprises only the two above families. The first structure of a fungal GH12 enzyme was provided shortly after, for ''Trichoderma reesei'' (formerly ''Hypocrea jecorina'') Cel12A <cite>Sandgren2001</cite>. To date structural models, both in the apo-form and in complex with oligosaccharides, are available for sixteen family members.
-From the different complex structures it can be seen that the substrate-binding groove of family GH12 enzymes harbours six binding sites, from -4 to +2, lined by numerous aromatic residues providing staking platforms for the individual pyranose units. This is consistent with the endoglucanase activity of these enzymes, exhibiting low catalytic efficiency towards short chain substrates <cite>Karlsson2002</cite>. The [[catalytic nucleophile]] Glu120 (S. lividans CelB2 numbering) and the [[ general acid/base]] Glu203 are in close proximity of an invariant aspartate, which might have an important role in catalysis and the constellation of this catalytic triad is reminiscent of the catalytic centre of family GH7 enzymes. Like in family [[GH7]] enzymes the acid/base residues of family GH12 members protonate syn to the pyranoside 0-5-C-1 bond.
+From the various complex structures it can be seen that the substrate-binding grooves of family GH12 enzymes harbour six binding sites, from -4 to +2, lined by numerous aromatic residues providing binding platforms for the individual pyranose units. This is consistent with the endoglucanase activity of these enzymes, exhibiting low catalytic efficiency towards short chain substrates <cite>Karlsson2002</cite>. The [[catalytic nucleophile]] Glu120 (''S. lividans'' CelB2 numbering) and the [[general acid/base]] Glu203 are in close proximity of an invariant aspartate, which might have an important role in catalysis. This catalytic triad is reminiscent of the catalytic centre of family GH7 enzymes. GH12 family members, like those of family [[GH7]], have the acid/base residue situated 'syn' to the pyranoside 0-5-C-1 bond, and are thus classified as syn-protonators.
 [[File:gh12_2.png|thumb|300px|right|'''Figure 2: Xyloglucanase XG12 from ''Bacillus licheniformis'' in complex with xyloglucan (PDB [{{PDBlink}}}2jen 2JEN]).'''  &alpha;-1,6-xylose substitutions are present at subsites -3, -2, +1 and +2.]]
-For xyloglucan specific enzymes of the family, structural and functional studies of xyloglucanase XG12 from ''Bacillus licheniformis'' <cite>Gloster2007</cite> and the xyloglucan specific endo-&beta;-1,4-glucanase XEG1 from ''Aspergillus aculeatus KSM 50'' <cite>Yoshizawa2012</cite> have shown that &alpha;-1,6-xylose substitutions can be tolerated at subsites -3, -2, +1 and +2 (Fig. 2). Certain GH12 enzymes exhibit &beta;-1,3-1,4-glucanase activity and the crystal structure of ''Humicola grisea'' Cel12A in complex with a mixed linkage oligosaccharide arising from a transglycosylation reaction revealed that the &beta;-1,3-linkage can be accommodated with ease between subsites -3 and -2 <cite>Sandgren2004</cite>. Speculations had been forwarded that a long loop crossing the substrate-binding groove at its reducing end, known as the “cord” and conserved in all [[clan]] GH-C members, might undergo conformational changes upon substrate binding <cite>Torronen1994</cite>, but the available GH12 complex structures support the view that the role of this loop is to deliver residues for substrate binding.
+For xyloglucan specific enzymes of the family, structural and functional studies of xyloglucanase XG12 from ''Bacillus licheniformis'' <cite>Gloster2007</cite> and the xyloglucan specific endo-&beta;-1,4-glucanase XEG1 from ''Aspergillus aculeatus KSM 50'' <cite>Yoshizawa2012</cite> have shown that &alpha;-1,6-xylose substitutions can be tolerated at subsites -3, -2, +1 and +2 (Fig. 2). Certain GH12 enzymes exhibit &beta;-1,3-1,4-glucanase activity and the crystal structure of ''Humicola grisea'' Cel12A in complex with a mixed linkage oligosaccharide arising from a transglycosylation reaction revealed that the &beta;-1,3-linkage can be accommodated with ease between subsites -3 and -2 <cite>Sandgren2004</cite>. It has been speculated that a long loop crossing the substrate-binding groove at its reducing end, known as the “cord” and conserved in all [[clan]] GH-C members, might undergo conformational changes upon substrate binding <cite>Torronen1994</cite>, but the available GH12 complex structures support the view that the role of this loop is to deliver residues for substrate binding.
-The crystal structure of Aspergillus aculeatus XEG1 in complex with the glycoside hydrolase inhibitory protein from carrot. EDGP, reveals an original mode of inhibition, distinct from the one observed previously for the inhibition of a family [[GH11]] xylanase by a wheat protein <cite>Payan2004</cite>. In the XEG1-GHIP complex the inhibitor mimics a xyloglucan substrate and inserts two conserved arginine residues into the active site, which establish salt bridges with the carboxylate groups of the catalytic center (Fig. 3) <cite>Yoshizawa2012</cite>).
+The crystal structure of ''Aspergillus aculeatus'' XEG1 in complex with the glycoside hydrolase inhibitory protein (GHIP) from carrot, EDGP, reveals a mode of inhibition distinct from that observed previously for the inhibition of a family [[GH11]] xylanase by a wheat protein <cite>Payan2004</cite>. In the XEG1-GHIP complex the inhibitor mimics a xyloglucan substrate and inserts two conserved arginine residues into the active site, which establish salt bridges with the carboxylate groups of the catalytic centre (Fig. 3) <cite>Yoshizawa2012</cite>).
 [[File:gh12_3.png|thumb|300px|right|'''Figure 3: Inhibition of ''Aspergillus aculeatus'' XEG1 by the glycoside hydrolase inhibitory protein from carrot. EDGP (PDB [{{PDBlink}}3vlb 3VLB]).''' Two conserved arginine residues of the inhibitor make salt bridges with the catalytic residues of XEG1. ''Colour coding:'' XEG1 in pink with carboxylate groups of the catalytic centre in purple; EDGP in lightblue with the conserved arginine residues in deepblue.]]
-Due to their commercial interest in bioprocessing applications, GH12 endo-&beta;-1,4-glucanases from ''Humicola grisea''  <cite>Sandgren2003a</cite>, ''Trichoderma reesei'' <cite>Sandgren2003a Sandgren2003b</cite>, ''Rhodothermus marinus'' <cite>Kapoor2008</cite>, ''Pyrococcus furiosus'' <cite>Kim2012</cite> and ''Thermotoga maritima'' <cite>Cheng2012</cite> have been extensively engineered and the structural studies of the resulting variants have provided valuable insight into structural features governing enzyme activity and stability.
+Due to commercial interest in their bioprocessing applications, GH12 endo-&beta;-1,4-glucanases from ''Humicola grisea''  <cite>Sandgren2003a</cite>, ''Trichoderma reesei'' <cite>Sandgren2003a Sandgren2003b</cite>, ''Rhodothermus marinus'' <cite>Kapoor2008</cite>, ''Pyrococcus furiosus'' <cite>Kim2012</cite> and ''Thermotoga maritima'' <cite>Cheng2012</cite> have been extensively engineered and the structural studies of the resulting variants have provided valuable insight into structural features governing enzyme activity and stability.
 == Family Firsts ==
-;First sterochemistry determination: ''Humicola insolens'' endoglucanase 3 by NMR  <cite>Schou1993</cite>.
+;First sterochemistry determination: ''Humicola insolens'' endoglucanase 3 by <sup>1<</sup>NMR  <cite>Schou1993</cite>.
-;First [[catalytic nucleophile]] identification: The catalytic nucleophile was first identified in ''Streptomyces lividans'' CelB2 by trapping of a glycosyl-enzyme intermediate followed by X-ray structure determination <cite>Sulzenbacher1999</cite> and peptide mapping via LC-MS/MS technologies <cite>Zechel1998</cite>.
+;First [[catalytic nucleophile]] identification: The catalytic nucleophile was first identified in ''Streptomyces lividans'' CelB2 by trapping of a glycosyl-enzyme intermediate and X-ray structure determination <cite>Sulzenbacher1999</cite> and peptide mapping using LC-MS/MS <cite>Zechel1998</cite>.
-;First [[general acid/base]] residue identification: inferred from homology with family [[GH11]] enzymes
+;First [[general acid/base]] residue identification: inferred from homology with family [[GH11]] enzymes.
-;First 3-D structure: The crystal structure of ''Streptomyces lividans'' CelB2 <cite>Sulzenbacher1997</cite>
+;First 3-D structure: The crystal structure of ''Streptomyces lividans'' CelB2 <cite>Sulzenbacher1997</cite>.
 == References ==

@@ Line 34: / Line 34: @@
 == Catalytic Residues ==
-The [[catalytic nucleophile]] and the [[general acid/base]] catalyst of GH12 members (previously known as "family H cellulases") was initialy predicted by sequence homology to the xylanase members of [[GH11]] <cite>Torronen1993</cite>, a glycoside hydrolase family where the [[catalytic nucleophile]] was first identified in the ''Bacillus circulans'' [[endo]]-xylanase through trapping of the 2-deoxy-2-fluoroxylobiosyl-enzyme [[intermediate]] and subsequent peptide mapping via LC-MS/MS technologies <cite>Miao1994</cite>. [[GH11]] and GH12 together form [[clan]] GH-C. The prediction of the [[catalytic nucleophile]] and the [[general acid/base]] of GH family 12 enzymes was later confirmed to be correct when the first three dimensional structure of a GH family 12 enzyme was determined, that of ''Streptomyces lividans'' CelB2 <cite>Sulzenbacher1997</cite>. The [[catalytic nucleophile]] was subsequentialy confirmed in ''Streptomyces lividans'' CelB2 to be Glu 120 by using the same labeling strategy used for detecting the [[catalytic nucleophile]] of family [[GH11]] <cite>Zechel1998</cite>.
+The [[catalytic nucleophile]] and the [[general acid/base]] catalyst of GH12 members (previously known as "family H cellulases" <cite>Gilkes1991 Torronen1993</cite>) was initialy predicted by sequence homology to the xylanase members of [[GH11]] <cite>Torronen1993</cite>, a glycoside hydrolase family where the [[catalytic nucleophile]] was first identified in the ''Bacillus circulans'' [[endo]]-xylanase through trapping of the 2-deoxy-2-fluoroxylobiosyl-enzyme [[intermediate]] and subsequent peptide mapping via LC-MS/MS technologies <cite>Miao1994</cite>. [[GH11]] and GH12 together form [[clan]] GH-C. The prediction of the [[catalytic nucleophile]] and the [[general acid/base]] of GH family 12 enzymes was later confirmed to be correct when the first three dimensional structure of a GH family 12 enzyme was determined, that of ''Streptomyces lividans'' CelB2 <cite>Sulzenbacher1997</cite>. The [[catalytic nucleophile]] was subsequentialy confirmed in ''Streptomyces lividans'' CelB2 to be Glu 120 by using the same labeling strategy used for detecting the [[catalytic nucleophile]] of family [[GH11]] <cite>Zechel1998</cite>.
 == Three-dimensional structures ==
@@ Line 87: / Line 87: @@
 #Sulzenbacher1999 pmid=10200171
 #Nielsen2002 Nielsen, RI. (2002) ''Microbial xyloglucan endotransglycosylase (XET)'' Patent [{{PatentLink}}US6448056 US6448056].
 </biblio>
 [[Category:Glycoside Hydrolase Families|GH012]]