Glycoside Hydrolase Family 81 - Revision history

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

2021-12-18T21:20:28Z

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

Al Boraston at 21:48, 18 August 2020

2020-08-18T21:48:40Z

Harry Brumer: /* Three-dimensional structures */

2020-08-07T16:08:48Z

Three-dimensional structures

Harry Brumer: /* Catalytic Residues */

2020-08-07T16:03:16Z

Catalytic Residues

Harry Brumer: /* Kinetics and Mechanism */

2020-08-07T16:00:47Z

Kinetics and Mechanism

Harry Brumer: /* Substrate specificities */

2020-08-07T16:00:34Z

Substrate specificities

Al Boraston at 23:44, 6 August 2020

2020-08-06T23:44:18Z

Julie Grondin at 17:04, 22 July 2020

2020-07-22T17:04:31Z

Julie Grondin at 17:03, 22 July 2020

2020-07-22T17:03:41Z

Julie Grondin at 17:02, 22 July 2020

2020-07-22T17:02:33Z

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 <!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
 {{CuratorApproved}}
-* [[Author]]: ^^^Julie Grondin^^^
+* [[Author]]: [[User:Julie Grondin|Julie Grondin]]
-* [[Responsible Curator]]:  ^^^Al Boraston^^^
+* [[Responsible Curator]]:  [[User:Al Boraston|Al Boraston]]
 ----

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 Primary sequence alignments of GH81 members reveal a number of highly conserved residues, including two glutamate residues and one aspartate residue, which are located in the active site cleft and are likely to serve as catalytic residues <cite>Martin-Cuadrado2008, McGrath2009, Zhou2013</cite>.
-Comprehensive mutagenesis experiments, azide rescue, structural analysis, and examination of the product conformational itinerary in ligand complexes show that one of the two glutamic acids (E542 in GH81 from ''Bacillus halodurans'' C-125) acts as the catalytic base by activating a catalytic water, and that the aspartic acid (D422 in ''Bh''GH81) acts as the catalytic acid  <cite>McGrath2009, Pluvinage2017</cite>. Mutagenesis studies in ''T. fusca'' indicate that mutation of the second glutamic acid (E546 in ''Bh''GH81) results in a dramatic reduction in activity  <cite>McGrath2009</cite>. Structural analysis of ''Bh''GH81 indicates that this residue is positioned during the catalytic cycle to interact with and stabilize a distorted product intermediate. The potential role of this residue as a base and in activating the catalytic water prior to substrate binding is presently unclear.
+Comprehensive mutagenesis experiments, azide rescue, structural analysis, and examination of the product conformational itinerary in ligand complexes show that one of the two glutamic acids (E542 in GH81 from ''Bacillus halodurans'' C-125) acts as the catalytic base by activating a catalytic water, and that the aspartic acid (D466 in ''Bh''GH81) acts as the catalytic acid  <cite>McGrath2009, Pluvinage2017</cite>. Mutagenesis studies in ''T. fusca'' indicate that mutation of the second glutamic acid (E546 in ''Bh''GH81) results in a dramatic reduction in activity  <cite>McGrath2009</cite>. Structural analysis of ''Bh''GH81 indicates that this residue is positioned during the catalytic cycle to interact with and stabilize a distorted product intermediate. The potential role of this residue as a base and in activating the catalytic water prior to substrate binding is presently unclear.
 == Three-dimensional structures ==

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 == Three-dimensional structures ==
 [[File:figure1_4k3a.png|400px|thumb|right|'''Figure 1. Structure of Lam81A from ''Rhizomucor meihei''''' ([{{PDBlink}}4K3A PDB ID 4K3A]). Domain A (blue) and Domain C (green) comprise the core of the enzyme, with domain B (orange) acting as a stabilizer. The proposed catalytic residues are shown as red sticks.]]
-GH81 are multimodular, although the composition of the domains can vary slightly. The first characterized structure, Lam81A from ''Rhizomucor miehei'' CAU432, comprises an N-terminal &beta;-sandwich domain (domain A), a small &alpha;/&beta; domain (domain B), and a C-terminal (&alpha;/&alpha;)<sub>6</sub> domain (domain C)  <cite>Zhou2013</cite>. Domains A and C form the core of the enzyme, which is likely stabilized by domain B. ('''Figure 1'''). This architecture is largely conserved in the GH81 from ''Bacillus halodurans'' C-125 (''Bh''GH81) <cite>Pluvinage2017</cite>, and the cellulosomal GH81 from ''Clostridium themocellum'' ATCC 27405 (''Ct''Lam81A) <cite>Kumar2018</cite>, however, the C-terminal domain is a [[CBM56]] in ''Bh''GH81 and a cellulosomal dockerin in ''Ct''Lam81A.
+GH81 members are multimodular, although the composition of the domains can vary slightly. The first characterized structure, Lam81A from ''Rhizomucor miehei'' CAU432, comprises an N-terminal &beta;-sandwich domain (domain A), a small &alpha;/&beta; domain (domain B), and a C-terminal (&alpha;/&alpha;)<sub>6</sub> domain (domain C)  <cite>Zhou2013</cite>. Domains A and C form the core of the enzyme, which is likely stabilized by domain B. ('''Figure 1'''). This architecture is largely conserved in the GH81 from ''Bacillus halodurans'' C-125 (''Bh''GH81) <cite>Pluvinage2017</cite>, and the cellulosomal GH81 from ''Clostridium themocellum'' ATCC 27405 (''Ct''Lam81A) <cite>Kumar2018</cite>, however, the C-terminal domain is a [[CBM56]] in ''Bh''GH81 and a cellulosomal dockerin in ''Ct''Lam81A.
-GH81 structures are unique among GHs and also differ from other characterized endo-&beta;(1,3)-glucanases in the PDB. As such, GH81 is not classified into any GH clan.
+[[File:Figure2_helical.PNG|400px|thumb|right|'''Figure 2. The structure of GH81 from ''Bacillus halodurans'' suggests that GH81 are capable of binding helical forms of &beta;-glucan''' ([{{PDBlink}}5T4G PDB ID 5T4G]). The triple helical structure of curdlan (beige, yellow, cyan) is shown, with the pitch (16Å) and spacing (5.3Å) between strands indicated. Figure from <cite>Pluvinage2017</cite>.]]
-[[File:Figure2_helical.PNG|400px|thumb|right|'''Figure 2. The structure of GH81 from ''Bacillus halodurans'' suggests that GH81 are capable of binding helical forms of &beta;-glucan''' ([{{PDBlink}}5T4G PDB ID 5T4G]). The triple helical structure of curdlan (beige, yellow, cyan) is shown, with the pitch (16Å) and spacing (5.3Å) between strands indicated. Figure from <cite>Pluvinage2017</cite>.]]
+GH81 tertiary structures are unique among GHs, including other characterized endo-&beta;(1,3)-glucanase families. As such, GH81 is not classified into any GH [[clan]].
 == Family Firsts ==

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 == Catalytic Residues ==
-Primary sequence alignments of GH81 reveal a number of highly conserved residues, including two glutamate residues and one aspartate residue which are located in the active site cleft and likely to serve as catalytic residues <cite>Martin-Cuadrado2008, McGrath2009, Zhou2013</cite>.
+Primary sequence alignments of GH81 members reveal a number of highly conserved residues, including two glutamate residues and one aspartate residue, which are located in the active site cleft and are likely to serve as catalytic residues <cite>Martin-Cuadrado2008, McGrath2009, Zhou2013</cite>.
-Comprehensive mutagenesis experiments, azide rescue, structural analysis, and examination of the product conformational itinerary in ligand complexes show that one of the two glutamic acids (E542 in GH81 from ''Bacillus halodurans'' C-125) acts as the catalytic base by activating a catalytic water, and that the aspartic acid (D422 in ''Bh''GH81) acts as the catalytic acid  <cite>McGrath2009, Pluvinage2017</cite>. Mutagenesis studies in T. fusca indicate that mutation of the second glutamic acid (E546 in ''Bh''GH81) results in a dramatic reduction in activity  <cite>McGrath2009</cite>. Structural analysis in ''B. halodurans'' indicates that this residue is positioned during the catalytic cycle to interact with and stabilize a distorted product intermediate. As such, the potential role of this residue as a base and in activating the catalytic water prior to substrate binding is presently unclear.
+Comprehensive mutagenesis experiments, azide rescue, structural analysis, and examination of the product conformational itinerary in ligand complexes show that one of the two glutamic acids (E542 in GH81 from ''Bacillus halodurans'' C-125) acts as the catalytic base by activating a catalytic water, and that the aspartic acid (D422 in ''Bh''GH81) acts as the catalytic acid  <cite>McGrath2009, Pluvinage2017</cite>. Mutagenesis studies in ''T. fusca'' indicate that mutation of the second glutamic acid (E546 in ''Bh''GH81) results in a dramatic reduction in activity  <cite>McGrath2009</cite>. Structural analysis of ''Bh''GH81 indicates that this residue is positioned during the catalytic cycle to interact with and stabilize a distorted product intermediate. The potential role of this residue as a base and in activating the catalytic water prior to substrate binding is presently unclear.
 == Three-dimensional structures ==

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 == Kinetics and Mechanism ==
-GH81 enzymes follow an [[inverting]] mechanism, first shown by <sup>1</sup>H-NMR during the hydrolysis of laminarin oligosaccharides <cite>Fliegmann2005</cite>, and laminarin <cite>McGrath2006</cite>, thus operating by a [[Glycoside_hydrolases#Inverting_glycoside_hydrolases|single-displacement mechanism]].
+GH81 enzymes follow an [[inverting]] mechanism, as first shown by <sup>1</sup>H-NMR during the hydrolysis of laminarin oligosaccharides <cite>Fliegmann2005</cite>, and laminarin <cite>McGrath2006</cite>, thus operating by a [[Glycoside_hydrolases#Inverting_glycoside_hydrolases|single-displacement mechanism]].
 == Catalytic Residues ==

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 == Substrate specificities ==
-GH81 family are endo-&beta;(1,3)-glucanases ([{{EClink}}3.2.1.39 EC 3.2.1.39]) with diverse physiological roles, such as plant biomass degradation, cell cycling, and enzymatic pathogen defense. They are mostly found in bacteria and fungi, and are particularly abundant in ''Saccharomyces'' and ''Streptomyces'' species. Activity has been demonstrated on laminarin <cite>Fontaine1997, McGrath2006, Martin-Cuadrado2008, Zhou2013, Pluvinage2017, Kumar2018</cite>, curdlan <cite>Fontaine1997, Martin-Cuadrado2008, Pluvinage2017, Kumar2018</cite>, and pachyman  <cite>McGrath2006, Pluvinage2017</cite>.
+GH81 members are endo-&beta;(1,3)-glucanases ([{{EClink}}3.2.1.39 EC 3.2.1.39]) with diverse physiological roles in, for example, plant biomass degradation, cell cycling, and pathogen defense. They are mostly found in bacteria and fungi, and are particularly abundant in ''Saccharomyces'' and ''Streptomyces'' species. Activity has been demonstrated on laminarin <cite>Fontaine1997, McGrath2006, Martin-Cuadrado2008, Zhou2013, Pluvinage2017, Kumar2018</cite>, curdlan <cite>Fontaine1997, Martin-Cuadrado2008, Pluvinage2017, Kumar2018</cite>, and pachyman  <cite>McGrath2006, Pluvinage2017</cite>.
 == Kinetics and Mechanism ==

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 == Family Firsts ==
 ;First stereochemistry determination: &beta;-glucan binding protein (GBP) from soybean (''Glycine max L.'') by <sup>1</sup>H-NMR <cite>Fliegmann2005</cite>.
-;First catalytic nucleophile identification: Lam81A from ''Thermobifida fusca'', by site-directed mutagenesis and azide rescue <cite>McGrath2009</cite>.
+;First catalytic nucleophile identification: Lam81A from ''Thermobifida fusca'', by site-directed mutagenesis and azide rescue <cite>McGrath2009</cite>, later confirmed by structural analysis <cite>Pluvinage2017</cite>.
-;First general acid/base residue identification: Lam81A from ''Thermobifida fusca'', by site-directed mutagenesis and azide rescue <cite>McGrath2009</cite>, later confirmed by structural analysis <cite>Pluvinage2017</cite>.
+;First general acid/base residue identification: Lam81A from ''Thermobifida fusca'', by site-directed mutagenesis <cite>McGrath2009</cite>, later confirmed by structural analysis <cite>Pluvinage2017</cite>.
 ;First 3-D structure: Lam81A from ''Rhizomucor miehei'' CAU432 <cite>Zhou2013</cite>.

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 [[File:Figure2_helical.PNG|400px|thumb|right|'''Figure 2. The structure of GH81 from ''Bacillus halodurans'' suggests that GH81 are capable of binding helical forms of &beta;-glucan''' ([{{PDBlink}}5T4G PDB ID 5T4G]). The triple helical structure of curdlan (beige, yellow, cyan) is shown, with the pitch (16Å) and spacing (5.3Å) between strands indicated. Figure from <cite>Pluvinage2017</cite>.]]
-Spanning domains A and C is a large cleft (10Å deep, 10Å wide, 70Å long), in which the proposed catalytic residues are located. Extensive co-crystallization of ''Bh''GH81 in complex with a range of laminarin oligosaccharides provides structural evidence for the ability of this enzyme for to generate a pool of oligosaccharide products <cite>Pluvinage2017</cite>. Notably, these structures clearly define catalytic and ancillary binding subsites, and reveal the ability of this enzyme to simultaneously bind oligosaccharides in these sites, proposing that GH81 bind and cleave helical forms of &beta;-1,3-glucans in an ''endo''-processive manner <cite>Pluvinage2017</cite> ('''Figure 2''').
+Spanning domains A and C is a large cleft (10Å deep, 10Å wide, 70Å long), in which the proposed catalytic residues are located. Extensive co-crystallization of ''Bh''GH81 in complex with a range of laminarin oligosaccharides provides structural evidence for the ability of this enzyme for to generate a pool of oligosaccharide products <cite>Pluvinage2017</cite>. Notably, these structures clearly define catalytic and ancillary binding subsites, and reveal the ability of this enzyme to simultaneously bind oligosaccharides in these sites. Thus, GH81 is likely to bind and cleave helical forms of &beta;-1,3-glucans in an ''endo''-processive manner <cite>Pluvinage2017</cite> ('''Figure 2''').
 == Family Firsts ==