Glycoside Hydrolase Family 52 - Revision history

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

2021-12-18T21:15:56Z

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

Joel Weadge: /* Substrate specificities */

2020-12-02T19:28:51Z

Substrate specificities

Harry Brumer: Increased figure size for legibility

2020-09-04T15:58:41Z

Increased figure size for legibility

Harry Brumer: /* Three-dimensional structures */

2020-09-04T15:34:44Z

Three-dimensional structures

Harry Brumer: /* Three-dimensional structures */

2020-09-04T15:33:07Z

Three-dimensional structures

Harry Brumer: /* Substrate specificities */

2020-09-04T15:30:54Z

Substrate specificities

Harry Brumer at 15:27, 4 September 2020

2020-09-04T15:27:27Z

Joel Weadge at 02:03, 4 September 2020

2020-09-04T02:03:06Z

Joel Weadge at 01:59, 4 September 2020

2020-09-04T01:59:01Z

Joel Weadge at 01:58, 4 September 2020

2020-09-04T01:58:01Z

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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^^^ and ^^^Brian Lowrance^^^
+* [[Author]]: [[User:Julie Grondin|Julie Grondin]] and [[User:Brian Lowrance|Brian Lowrance]]
-* [[Responsible Curator]]: ^^^Joel Weadge^^^
+* [[Responsible Curator]]: [[User:Joel Weadge|Joel Weadge]]
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 == Substrate specificities ==
-The GH52 enzymes are often isolated from various mesophilic and thermophilic bacteria, which has led to a demonstrated high thermostability within this family. The enzymes are generally monospecific, functioning as ''exo''-&beta;-xylosidases (EC [{{EClink}}3.2.1.37 3.2.1.37]) that cleave the terminal xylose residues from the non-reducing end of artificial xylosides and xylooligosaccharides (e.g., ''p''NP-β-D-xylopyranoside <cite>Bravman2001, Espina2014</cite>, xylobiose <cite>Espina2014</cite>, and xylotriose <cite>Espina2014</cite>). Low levels of &alpha;-L-arabinofuranoside activity has also been observed within members of the GH52 family <cite>Suzuki2014, Bravman2003a</cite>, which is similar to the specificity noted for [[GH13]] and [[GH54]] for &beta;-xylooligosaccharides and &alpha;-L-arabinofuranosides. The specificity for these substrates is likely due to similarities in orientation of hydroxyls and glycosidic bonds of the substrate within the active site <cite>Lee2007, Utt1991</cite>. Under certain conditions, some enzymes in the family have also exhibited weak transglycosylation activity, a phenomenon that has also been infrequently observed in other [[glycoside hydrolase]] <cite>Romero2019</cite>. The plasticity of the active site of some GH52 members has been further explored through site-directed mutagenesis, where introduction of xylanase activity <cite>Huang2014</cite> and transition from a [[glycoside hydrolase]] to a glycosynthase <cite>Dann2014</cite> has been achieved.
+The GH52 enzymes are often isolated from various mesophilic and thermophilic bacteria, which has led to a demonstrated high thermostability within this family. The enzymes are generally monospecific, functioning as ''exo''-&beta;-xylosidases (EC [{{EClink}}3.2.1.37 3.2.1.37]) that cleave the terminal xylose residues from the non-reducing end of artificial xylosides and xylooligosaccharides (e.g., ''p''NP-β-D-xylopyranoside <cite>Bravman2001, Espina2014</cite>, xylobiose <cite>Espina2014</cite>, and xylotriose <cite>Espina2014</cite>). Low levels of &alpha;-L-arabinofuranoside activity has also been observed within members of the GH52 family <cite>Suzuki2014, Bravman2003a</cite>, which is similar to the specificity noted for [[GH13]] and [[GH54]] for &beta;-xylooligosaccharides and &alpha;-L-arabinofuranosides. The specificity for these substrates is likely due to similarities in orientation of hydroxyls and glycosidic bonds of the substrate within the active site <cite>Lee2007, Utt1991</cite>. Under certain conditions, some enzymes in the family have also exhibited weak transglycosylation activity, a phenomenon that has also been infrequently observed in other [[glycoside hydrolase]]s <cite>Romero2019</cite>. The plasticity of the active site of some GH52 members has been further explored through site-directed mutagenesis, where introduction of xylanase activity <cite>Huang2014</cite> and transition from a [[glycoside hydrolase]] to a glycosynthase <cite>Dann2014</cite> has been achieved.
 == Kinetics and Mechanism ==

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 == Three-dimensional structures ==
-[[File:Figure1_dimer.PNG|300px|thumb|right|'''Figure 1. The dimeric structure of GH52 from ''Geobacillus thermoglucosidasius'' in complex with xylobiose (orange)([{{PDBlink}}4C1P PDB ID 4C1P]).''' The active site is enclosed by residues from both monomers, restricting this enzyme to ''exo''-hydrolysis via steric hindrance of the catalytic site. Figure from <cite>Espina2014</cite>.]]
+[[File:Figure1_dimer.PNG|400px|thumb|right|'''Figure 1. The dimeric structure of GH52 from ''Geobacillus thermoglucosidasius'' in complex with xylobiose (orange)([{{PDBlink}}4C1P PDB ID 4C1P]).''' The active site is enclosed by residues from both monomers, restricting this enzyme to ''exo''-hydrolysis via steric hindrance of the catalytic site. Figure from <cite>Espina2014</cite>.]]
 Representative structures of GH52 glycoside hydrolases have been solved, XynB2 from ''B. stearothermophilus'' T-6 ([{{PDBlink}}4RHH PDB ID 4RHH]) and GT2_24_00240 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]; [{{PDBlink}}4C1O PDB ID 4C1O]). These enzymes have folds comprised of an N-terminal β-sandwich domain and a C-terminal (&alpha;/&alpha;)<sup>6</sup> barrel domain (Figure 1) that has led to their classification into [[Clan]] GH-O, together with [[GH116]]. The ''exo''-acting mode-of-action of GH52's is reflected in the topology of the active site. The enzymes act as dimers in solution <cite>Bravman2001, Espina2014</cite>, with interactions between monomers of the GH52 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]) forming a deep pocket to enclose and distort the non-reducing end xylose into a high-energy <sup>4</sup>H<sub>3</sub> half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site <cite>Espina2014</cite>. Furthermore, the structure of the active site also allosterically inhibits access to negative subsites beyond the -1 site. This permits interaction with only a single xylosyl residue in the negative subsites and thus hydrolysis yields a lone xylose molecule. In summary, this mechanism promotes strict ''exo''-&beta;-xylosidase activity, while inhibiting activity on large polymers, such as xylan.

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 [[File:Figure1_dimer.PNG|300px|thumb|right|'''Figure 1. The dimeric structure of GH52 from ''Geobacillus thermoglucosidasius'' in complex with xylobiose (orange)([{{PDBlink}}4C1P PDB ID 4C1P]).''' The active site is enclosed by residues from both monomers, restricting this enzyme to ''exo''-hydrolysis via steric hindrance of the catalytic site. Figure from <cite>Espina2014</cite>.]]
-Representative structures of GH52 glycoside hydrolases have been solved, XynB2 from ''B. stearothermophilus'' T-6 ([{{PDBlink}}4RHH PDB ID 4RHH]) and GT2_24_00240 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]; [{{PDBlink}}4C1O PDB ID 4C1O]). These enzymes have folds comprised of an N-terminal β-sandwich domain and a C-terminal (&alpha;/&alpha;)<sup>6</sup> barrel domain (Figure 1) that has led to their classification into the GH-''O'' [[clan]], which is similar to that noted for the [[GH116]] family. The ''exo''-acting mode-of-action of GH52's is reflected in the topology of the active site. The enzymes act as dimers in solution <cite>Bravman2001, Espina2014</cite>, with interactions between monomers of the GH52 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]) forming a deep pocket to enclose and distort the non-reducing end xylose into a high-energy <sup>4</sup>H<sub>3</sub> half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site <cite>Espina2014</cite>. Furthermore, the structure of the active site also allosterically inhibits access to negative subsites beyond the -1 site. This permits interaction with only a single xylosyl residue in the negative subsites and thus hydrolysis yields a lone xylose molecule. In summary, this mechanism promotes strict ''exo''-&beta;-xylosidase activity, while inhibiting activity on large polymers, such as xylan.
+Representative structures of GH52 glycoside hydrolases have been solved, XynB2 from ''B. stearothermophilus'' T-6 ([{{PDBlink}}4RHH PDB ID 4RHH]) and GT2_24_00240 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]; [{{PDBlink}}4C1O PDB ID 4C1O]). These enzymes have folds comprised of an N-terminal β-sandwich domain and a C-terminal (&alpha;/&alpha;)<sup>6</sup> barrel domain (Figure 1) that has led to their classification into [[Clan]] GH-O, together with [[GH116]]. The ''exo''-acting mode-of-action of GH52's is reflected in the topology of the active site. The enzymes act as dimers in solution <cite>Bravman2001, Espina2014</cite>, with interactions between monomers of the GH52 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]) forming a deep pocket to enclose and distort the non-reducing end xylose into a high-energy <sup>4</sup>H<sub>3</sub> half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site <cite>Espina2014</cite>. Furthermore, the structure of the active site also allosterically inhibits access to negative subsites beyond the -1 site. This permits interaction with only a single xylosyl residue in the negative subsites and thus hydrolysis yields a lone xylose molecule. In summary, this mechanism promotes strict ''exo''-&beta;-xylosidase activity, while inhibiting activity on large polymers, such as xylan.
 == Family Firsts ==

@@ Line 40: / Line 40: @@
 [[File:Figure1_dimer.PNG|300px|thumb|right|'''Figure 1. The dimeric structure of GH52 from ''Geobacillus thermoglucosidasius'' in complex with xylobiose (orange)([{{PDBlink}}4C1P PDB ID 4C1P]).''' The active site is enclosed by residues from both monomers, restricting this enzyme to ''exo''-hydrolysis via steric hindrance of the catalytic site. Figure from <cite>Espina2014</cite>.]]
-The structures of two GH52 glycoside hydrolases have been solved, XynB2 from ''B. stearothermophilus'' T-6 ([{{PDBlink}}4RHH PDB ID 4RHH]) and GT2_24_00240 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]; [{{PDBlink}}4C1O PDB ID 4C1O]). These enzymes have folds comprised of an N-terminal β-sandwich domain and a C-terminal (&alpha;/&alpha;)<sup>6</sup> barrel domain (See Fig. 1) that has led to their classification into the GH-''O'' clan, which is similar to that noted for the GH116 family. The ''exo''-acting mode-of-action of GH52's is reflected in the topology of the active site. The enzymes act as dimers in solution <cite>Bravman2001, Espina2014</cite>, with interactions between monomers of the GH52 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]) forming a deep pocket to enclose and distort the non-reducing end xylose into a high-energy <sup>4</sup>H<sub>3</sub> half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site <cite>Espina2014</cite>. Furthermore, the structure of the active site also allosterically inhibits access to negative subsites beyond the -1 site. This permits interaction with only a single xylosyl residue in the negative subsites and thus hydrolysis yields a lone xylose molecule. In summary, this mechanism promotes strict ''exo''-&beta;-xylosidase activity, while inhibiting activity on large polymers, such as xylan.
+Representative structures of GH52 glycoside hydrolases have been solved, XynB2 from ''B. stearothermophilus'' T-6 ([{{PDBlink}}4RHH PDB ID 4RHH]) and GT2_24_00240 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]; [{{PDBlink}}4C1O PDB ID 4C1O]). These enzymes have folds comprised of an N-terminal β-sandwich domain and a C-terminal (&alpha;/&alpha;)<sup>6</sup> barrel domain (Figure 1) that has led to their classification into the GH-''O'' [[clan]], which is similar to that noted for the [[GH116]] family. The ''exo''-acting mode-of-action of GH52's is reflected in the topology of the active site. The enzymes act as dimers in solution <cite>Bravman2001, Espina2014</cite>, with interactions between monomers of the GH52 from ''G. thermoglucosidasius'' ([{{PDBlink}}4C1P PDB ID 4C1P]) forming a deep pocket to enclose and distort the non-reducing end xylose into a high-energy <sup>4</sup>H<sub>3</sub> half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site <cite>Espina2014</cite>. Furthermore, the structure of the active site also allosterically inhibits access to negative subsites beyond the -1 site. This permits interaction with only a single xylosyl residue in the negative subsites and thus hydrolysis yields a lone xylose molecule. In summary, this mechanism promotes strict ''exo''-&beta;-xylosidase activity, while inhibiting activity on large polymers, such as xylan.
 == Family Firsts ==

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 == Kinetics and Mechanism ==
-Retention of stereochemistry has been observed in GH52 &beta;-xylosidases, which is characteristic of a classical [[Koshland double-displacement mechanism]]  <cite>Koshland1953</cite>. This was first determined by Bravmen and coworkers using <sup>1</sup>H-NMR to analyze the breakdown products of ''p''NP-β-D-xylopyranoside by XynB2, a &beta;-xylosidase from ''Bacillus stearothermophilus'' T-6 <cite>Bravman2001</cite>. Further detailed analysis within this family was published in 2003 on the ''B. stearothermophilus'' XynB2 enzyme, which contained pH dependence studies (enzymatic catalysis is dependent on ionizable residues E335 and D495, with free enzyme experimental pKa values of 4.2 and 7.3, respectively) and kinetic analyses (''p''NP-xylobiose ''k''<sub>cat</sub>/''K''<sub>m</sub> of 140 s<sup>-1</sup>mM<sup>-1</sup>; xylobiose and xylotriose ''K''<sub>m</sub> values of 17.1x10<sup>4</sup> M<sup>-1</sup> and 9.6x10<sup>4</sup> M-<sup>1</sup>, respectively) <cite>Bravman2003a</cite>.
+Retention of stereochemistry has been observed in GH52 &beta;-xylosidases, which is characteristic of a classical [[Koshland double-displacement mechanism]]  <cite>Koshland1953</cite>. This was first determined by Bravmen and coworkers using <sup>1</sup>H-NMR to analyze the breakdown products of ''p''NP-β-D-xylopyranoside by XynB2, a &beta;-xylosidase from ''Bacillus stearothermophilus'' T-6 <cite>Bravman2001</cite>. Further detailed analysis within this family was published in 2003 on the ''B. stearothermophilus'' XynB2 enzyme, which contained pH dependence studies (enzymatic catalysis is dependent on ionizable residues E335 and D495, with free enzyme experimental pKa values of 4.2 and 7.3, respectively) and kinetic analyses (''p''NP-xylobiose ''k''<sub>cat</sub>/''K''<sub>M</sub> of 140 s<sup>-1</sup>mM<sup>-1</sup>; xylobiose and xylotriose ''K''<sub>M</sub> values of 17.1x10<sup>4</sup> M<sup>-1</sup> and 9.6x10<sup>4</sup> M-<sup>1</sup>, respectively) <cite>Bravman2003a</cite>.
 == Catalytic Residues ==