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Glycoside Hydrolase Family 36 - Revision history
2024-03-29T11:23:36Z
Revision history for this page on the wiki
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Harry Brumer: Text replacement - "\^\^\^(.*)\^\^\^" to "$1"
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<p>Text replacement - "\^\^\^(.*)\^\^\^" to "<a href="/index.php?title=User:$1&action=edit&redlink=1" class="new" title="User:$1 (page does not exist)">$1</a>"</p>
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Harry Brumer
https://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_36&diff=9363&oldid=prev
Harry Brumer at 03:46, 6 October 2013
2013-10-06T03:46:22Z
<p></p>
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<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Kinetics and Mechanism ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Kinetics and Mechanism ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Family GH36 &alpha;-galactosidases are anomeric configuration-[[retaining]] enzymes, as first shown by NMR studies on the &alpha;-galactosidase GalA from ''Thermotoga maritima'' <cite>Comfort2007</cite>. Correspondingly, GH36 enzymes use a classical [[Koshland double-displacement mechanism]] <cite>Sinnott1990</cite>, like their [[<del class="diffchange diffchange-inline">Glycoside Hydrolase Family GH27 (GH27)|</del>GH27]] relatives in Clan GH-D. This mechanism involves the formation of a covalent glycosyl-enzyme [[intermediate]] <cite>Vocadlo2001</cite> that partitions predominantly to water in hydrolytic enzymes and to saccharide acceptor substrates in transglycosylating enzymes, such as stachyose and raffinose synthases.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Family GH36 &alpha;-galactosidases are anomeric configuration-[[retaining]] enzymes, as first shown by NMR studies on the &alpha;-galactosidase GalA from ''Thermotoga maritima'' <cite>Comfort2007</cite>. Correspondingly, GH36 enzymes use a classical [[Koshland double-displacement mechanism]] <cite>Sinnott1990</cite>, like their [[GH27]] relatives in Clan GH-D. This mechanism involves the formation of a covalent glycosyl-enzyme [[intermediate]] <cite>Vocadlo2001</cite> that partitions predominantly to water in hydrolytic enzymes and to saccharide acceptor substrates in transglycosylating enzymes, such as stachyose and raffinose synthases.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Catalytic Residues ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Catalytic Residues ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of archaeal GH36 &alpha;-galactosidases within [[Sequence-based classification of glycoside hydrolases|Clan]] GH-D originally highlighted likely candidates for the [[catalytic nucleophile]] and [[general acid/base]] residues in this family, based on protein sequence similarity with those identified in [[<del class="diffchange diffchange-inline">Glycoside Hydrolase Family GH27 (GH27)|</del>GH27]] <cite>Brouns2006</cite>. Mutagenesis of the corresponding residues in ''Sulfolobus solfataricus'' &alpha;-galactosidase GalS dramatically reduced enzyme activity: the D367G ([[catalytic nucleophile]]) and D425G ([[general acid/base]]) mutant had <1 x 10<sup>–3</sup> and 5 x 10<sup>–3</sup> lower activity than the wild type enzyme when assayed against ''p''-nitrophenyl &alpha;-D-galactopyranoside <cite>Brouns2006</cite>. Rescue of the catalytic function of both enzyme mutants was unsuccessful with both azide and formate anions <cite>Brouns2006</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of archaeal GH36 &alpha;-galactosidases within [[Sequence-based classification of glycoside hydrolases|Clan]] GH-D originally highlighted likely candidates for the [[catalytic nucleophile]] and [[general acid/base]] residues in this family, based on protein sequence similarity with those identified in [[GH27]] <cite>Brouns2006</cite>. Mutagenesis of the corresponding residues in ''Sulfolobus solfataricus'' &alpha;-galactosidase GalS dramatically reduced enzyme activity: the D367G ([[catalytic nucleophile]]) and D425G ([[general acid/base]]) mutant had <1 x 10<sup>–3</sup> and 5 x 10<sup>–3</sup> lower activity than the wild type enzyme when assayed against ''p''-nitrophenyl &alpha;-D-galactopyranoside <cite>Brouns2006</cite>. Rescue of the catalytic function of both enzyme mutants was unsuccessful with both azide and formate anions <cite>Brouns2006</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>The identities of the catalytic residues in GH36 were also confirmed in the ''Thermotoga maritima'' &alpha;-galactosidase GalA, guided by structural homology with [[<del class="diffchange diffchange-inline">Glycoside Hydrolase Family GH27 (GH27)|</del>GH27]] enzymes <cite>Comfort2007</cite>. Site-directed mutation of Asp327 to Gly yielded a variant that had a 200-800-fold lower rate on aryl galactosides compared with the WT enzyme. Addition of azide was shown to rescue the ability of the enzyme to cleave ''p''-nitrophenyl &alpha;-D-galactopyranoside and resulted in formation of &beta;-galactopyranosyl azide, confirming Asp327 as the [[catalytic nucleophile]]. Mutation of the predicted [[general acid/base]] residue, Asp387, to Gly reduced activity 1500-fold on ''p''-nitrophenyl &alpha;-D-galactopyranoside, while addition of azide resulted in formation of &alpha;-galactopyranosyl azide by nucleophilic attack on the &beta;-linked glycosyl enzyme [[intermediate]].</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>The identities of the catalytic residues in GH36 were also confirmed in the ''Thermotoga maritima'' &alpha;-galactosidase GalA, guided by structural homology with [[GH27]] enzymes <cite>Comfort2007</cite>. Site-directed mutation of Asp327 to Gly yielded a variant that had a 200-800-fold lower rate on aryl galactosides compared with the WT enzyme. Addition of azide was shown to rescue the ability of the enzyme to cleave ''p''-nitrophenyl &alpha;-D-galactopyranoside and resulted in formation of &beta;-galactopyranosyl azide, confirming Asp327 as the [[catalytic nucleophile]]. Mutation of the predicted [[general acid/base]] residue, Asp387, to Gly reduced activity 1500-fold on ''p''-nitrophenyl &alpha;-D-galactopyranoside, while addition of azide resulted in formation of &alpha;-galactopyranosyl azide by nucleophilic attack on the &beta;-linked glycosyl enzyme [[intermediate]].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Three-dimensional structures ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Three-dimensional structures ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>In June 2005, the first three-dimensional structural coordinates for a member of this family, &alpha;-galactosidase TmGalA from ''Thermotoga maritima'', were deposited by the Joint Center For Structural Genomics (JCSG) (X-ray, 2.34 Å, [<del class="diffchange diffchange-inline">http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=</del>1zy9 PDB 1zy9]) <cite>Lesley2002</cite>. Subsequent analysis of this data in the context of existing structures of [[GH27]] enzymes, revealed homologous active site residues, including two key catalytic Asp residues <cite>Comfort2007 Ernst2006</cite> and a number of conserved substrate-binding residues <cite>Comfort2007</cite>. The active sites of both [[GH27]] and GH36 enzymes are presented by (&alpha;/&beta;)<sub>8</sub> (TIM) barrel domains. Both GH36 and [[GH27]] enzymes contain a C-terminal &beta;-sheet domain of unknown function, although this domain is structurally different and more disordered in TmGalA compared with the homologous domain in [[GH27]] (e.g., ''Oryza sativa'' &alpha;-galactosidase, [{{PDBlink}}1uas PDB 1uas]). Notably, an extra N-terminal, primarily &beta;-sheet domain, which is not found in [[<del class="diffchange diffchange-inline">Glycoside Hydrolase Family GH27 (GH27)|</del>GH27]] enzymes, contributes a key substrate-binding residue (Trp65) to the active site of TmGalA (Trp65, replacing Trp164 in the ''O. sativa'' enzyme). Another notable active site substitution, this time within the TIM barrel itself, is the replacement of the aromatic residues Phe328 and Trp 291 in TmGalA with Ser102 and the Cys101-Cys132 disulfide in the ''O. sativa'' enzyme <cite>Comfort2007</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>In June 2005, the first three-dimensional structural coordinates for a member of this family, &alpha;-galactosidase TmGalA from ''Thermotoga maritima'', were deposited by the Joint Center For Structural Genomics (JCSG) (X-ray, 2.34 Å, [<ins class="diffchange diffchange-inline">{{PDBlink}}</ins>1zy9 PDB 1zy9]) <cite>Lesley2002</cite>. Subsequent analysis of this data in the context of existing structures of [[GH27]] enzymes, revealed homologous active site residues, including two key catalytic Asp residues <cite>Comfort2007 Ernst2006</cite> and a number of conserved substrate-binding residues <cite>Comfort2007</cite>. The active sites of both [[GH27]] and GH36 enzymes are presented by (&alpha;/&beta;)<sub>8</sub> (TIM) barrel domains. Both GH36 and [[GH27]] enzymes contain a C-terminal &beta;-sheet domain of unknown function, although this domain is structurally different and more disordered in TmGalA compared with the homologous domain in [[GH27]] (e.g., ''Oryza sativa'' &alpha;-galactosidase, [{{PDBlink}}1uas PDB 1uas]). Notably, an extra N-terminal, primarily &beta;-sheet domain, which is not found in [[GH27]] enzymes, contributes a key substrate-binding residue (Trp65) to the active site of TmGalA (Trp65, replacing Trp164 in the ''O. sativa'' enzyme). Another notable active site substitution, this time within the TIM barrel itself, is the replacement of the aromatic residues Phe328 and Trp 291 in TmGalA with Ser102 and the Cys101-Cys132 disulfide in the ''O. sativa'' enzyme <cite>Comfort2007</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of Clan GH-D, originally comprised only of GH36 and [[GH27]], has indicated that archaeal GH36 enzymes are more closely related to plant "alkaline" &alpha;-galactosidases, raffinose synthases, and stachyose synthases than to bacterial and fungal members. Thermophilic bacterial sequences, such as TmGalA, appear to form a divergent cluster within this latter subgroup <cite>Brouns2006</cite>. Notably, crystallographic studies on an archeal member of [[GH31]] indicated the structural similarity with both GH36 and [[GH27]], resulting in the unification of these three families into Clan GH-D <cite>Ernst2006</cite>.</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of Clan GH-D, originally comprised only of GH36 and [[GH27]], has indicated that archaeal GH36 enzymes are more closely related to plant "alkaline" &alpha;-galactosidases, raffinose synthases, and stachyose synthases than to bacterial and fungal members. Thermophilic bacterial sequences, such as TmGalA, appear to form a divergent cluster within this latter subgroup <cite>Brouns2006</cite>. Notably, crystallographic studies on an archeal member of [[GH31]] indicated the structural similarity with both GH36 and [[GH27]], resulting in the unification of these three families into Clan GH-D <cite>Ernst2006</cite>.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Family Firsts ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Family Firsts ==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>;First sterochemistry determination: ''Thermotoga maritima'' &alpha;-galactosidase, by NMR <cite>Comfort2007</cite>.</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>;First sterochemistry determination: ''Thermotoga maritima'' &alpha;-galactosidase, by NMR <cite>Comfort2007</cite>.</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>;First [[catalytic nucleophile]] identification: ''Sulfolobus solfataricus'' &alpha;-galactosidase GalS, by sequence homology with [[<del class="diffchange diffchange-inline">Glycoside Hydrolase Family GH27 (GH27)|</del>GH27]] enzymes and mutagenesis <cite>Brouns2006</cite>. Subsequently confirmed in ''Thermotoga maritima'' &alpha;-galactosidase by structural homology, mutagenesis, and azide rescue <cite>Comfort2007</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>;First [[catalytic nucleophile]] identification: ''Sulfolobus solfataricus'' &alpha;-galactosidase GalS, by sequence homology with [[GH27]] enzymes and mutagenesis <cite>Brouns2006</cite>. Subsequently confirmed in ''Thermotoga maritima'' &alpha;-galactosidase by structural homology, mutagenesis, and azide rescue <cite>Comfort2007</cite>.</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>;First [[general acid/base]] residue identification: ''Sulfolobus solfataricus'' &alpha;-galactosidase GalS, by sequence homology with [[<del class="diffchange diffchange-inline">Glycoside Hydrolase Family GH27 (GH27)|</del>GH27]] enzymes and mutagenesis <cite>Brouns2006</cite>. Subsequently confirmed in ''Thermotoga maritima'' &alpha;-galactosidase by structural homology, mutagenesis, and azide rescue <cite>Comfort2007</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>;First [[general acid/base]] residue identification: ''Sulfolobus solfataricus'' &alpha;-galactosidase GalS, by sequence homology with [[GH27]] enzymes and mutagenesis <cite>Brouns2006</cite>. Subsequently confirmed in ''Thermotoga maritima'' &alpha;-galactosidase by structural homology, mutagenesis, and azide rescue <cite>Comfort2007</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>;First 3-D structure: ''Thermotoga maritima'' &alpha;-galactosidase by X-ray crystallography. Coordinates ([{{PDBlink}}1zy9 PDB 1zy9]) deposited in 2005 as part of a high-throughput functional genomics project <cite>Lesley2002</cite>, structural analyses published by other groups in 2006 and 2007 <cite>Comfort2007 Ernst2006</cite>.</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>;First 3-D structure: ''Thermotoga maritima'' &alpha;-galactosidase by X-ray crystallography. Coordinates ([{{PDBlink}}1zy9 PDB 1zy9]) deposited in 2005 as part of a high-throughput functional genomics project <cite>Lesley2002</cite>, structural analyses published by other groups in 2006 and 2007 <cite>Comfort2007 Ernst2006</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
</table>
Harry Brumer
https://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_36&diff=9362&oldid=prev
Harry Brumer: /* Three-dimensional structures */
2013-10-06T03:42:20Z
<p><span dir="auto"><span class="autocomment">Three-dimensional structures</span></span></p>
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 03:42, 6 October 2013</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>In June 2005, the first three-dimensional structural coordinates for a member of this family, &alpha;-galactosidase TmGalA from ''Thermotoga maritima'', were deposited by the Joint Center For Structural Genomics (JCSG) (X-ray, 2.34 Å, [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1zy9 PDB 1zy9]) <cite>Lesley2002</cite>. Subsequent analysis of this data in the context of existing structures of [[GH27]] enzymes, revealed homologous active site residues, including two key catalytic Asp residues <cite>Comfort2007 Ernst2006</cite> and a number of conserved substrate-binding residues <cite>Comfort2007</cite>. The active sites of both [[GH27]] and GH36 enzymes are presented by (&alpha;/&beta;)<sub>8</sub> (TIM) barrel domains. Both GH36 and [[GH27]] enzymes contain a C-terminal &beta;-sheet domain of unknown function, although this domain is structurally different and more disordered in TmGalA compared with the homologous domain in [[GH27]] (e.g., ''Oryza sativa'' &alpha;-galactosidase, [{{PDBlink}}1uas PDB 1uas]). Notably, an extra N-terminal, primarily &beta;-sheet domain, which is not found in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes, contributes a key substrate-binding residue (Trp65) to the active site of TmGalA (Trp65, replacing Trp164 in the ''O. sativa'' enzyme). Another notable active site substitution, this time within the TIM barrel itself, is the replacement of the aromatic residues Phe328 and Trp 291 in TmGalA with Ser102 and the Cys101-Cys132 disulfide in the ''O. sativa'' enzyme <cite>Comfort2007</cite>.</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>In June 2005, the first three-dimensional structural coordinates for a member of this family, &alpha;-galactosidase TmGalA from ''Thermotoga maritima'', were deposited by the Joint Center For Structural Genomics (JCSG) (X-ray, 2.34 Å, [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1zy9 PDB 1zy9]) <cite>Lesley2002</cite>. Subsequent analysis of this data in the context of existing structures of [[GH27]] enzymes, revealed homologous active site residues, including two key catalytic Asp residues <cite>Comfort2007 Ernst2006</cite> and a number of conserved substrate-binding residues <cite>Comfort2007</cite>. The active sites of both [[GH27]] and GH36 enzymes are presented by (&alpha;/&beta;)<sub>8</sub> (TIM) barrel domains. Both GH36 and [[GH27]] enzymes contain a C-terminal &beta;-sheet domain of unknown function, although this domain is structurally different and more disordered in TmGalA compared with the homologous domain in [[GH27]] (e.g., ''Oryza sativa'' &alpha;-galactosidase, [{{PDBlink}}1uas PDB 1uas]). Notably, an extra N-terminal, primarily &beta;-sheet domain, which is not found in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes, contributes a key substrate-binding residue (Trp65) to the active site of TmGalA (Trp65, replacing Trp164 in the ''O. sativa'' enzyme). Another notable active site substitution, this time within the TIM barrel itself, is the replacement of the aromatic residues Phe328 and Trp 291 in TmGalA with Ser102 and the Cys101-Cys132 disulfide in the ''O. sativa'' enzyme <cite>Comfort2007</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of Clan GH-D, comprised of GH36 and [[<del class="diffchange diffchange-inline">Glycoside Hydrolase Family GH27 (GH27)|</del>GH27]], has indicated that archaeal GH36 enzymes are more closely related to plant "alkaline" &alpha;-galactosidases, raffinose synthases, and stachyose synthases than to bacterial and fungal members. Thermophilic bacterial sequences, such as TmGalA, appear to form a divergent cluster within this latter subgroup <cite>Brouns2006</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of Clan GH-D, <ins class="diffchange diffchange-inline">originally </ins>comprised <ins class="diffchange diffchange-inline">only </ins>of GH36 and [[GH27]], has indicated that archaeal GH36 enzymes are more closely related to plant "alkaline" &alpha;-galactosidases, raffinose synthases, and stachyose synthases than to bacterial and fungal members. Thermophilic bacterial sequences, such as TmGalA, appear to form a divergent cluster within this latter subgroup <cite>Brouns2006<ins class="diffchange diffchange-inline"></cite>. Notably, crystallographic studies on an archeal member of [[GH31]] indicated the structural similarity with both GH36 and [[GH27]], resulting in the unification of these three families into Clan GH-D <cite>Ernst2006</ins></cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Family Firsts ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Family Firsts ==</div></td></tr>
</table>
Harry Brumer
https://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_36&diff=7510&oldid=prev
Harry Brumer: updated CAZyDBlink
2012-09-10T16:36:02Z
<p>updated CAZyDBlink</p>
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 16:36, 10 September 2012</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>|-</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>|-</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>| colspan="2" |<del class="diffchange diffchange-inline">http://www.cazy.org/fam/</del>GH36.html</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>| colspan="2" |<ins class="diffchange diffchange-inline">{{CAZyDBlink}}</ins>GH36.html</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>|}</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>|}</div></td></tr>
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</table>
Harry Brumer
https://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_36&diff=6889&oldid=prev
Spencer Williams at 03:05, 14 June 2011
2011-06-14T03:05:44Z
<p></p>
<table class="diff diff-contentalign-left diff-editfont-monospace" data-mw="interface">
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 03:05, 14 June 2011</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Substrate specificities ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Substrate specificities ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>[[Glycoside hydrolases]] of family 36 exhibit alpha-galactosidase and alpha-''N''-acetylgalactosaminidase activity, which has been demonstrated in archaeal, bacterial, and eukaryotic members of this family. Additionally, certain plant members of this family possess stachyose synthase or raffinose synthase activity.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>[[Glycoside hydrolases]] of family 36 exhibit <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase and <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-''N''-acetylgalactosaminidase activity, which has been demonstrated in archaeal, bacterial, and eukaryotic members of this family. Additionally, certain plant members of this family possess stachyose synthase or raffinose synthase activity.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Kinetics and Mechanism ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Kinetics and Mechanism ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Family GH36 alpha-galactosidases are anomeric configuration-[[retaining]] enzymes, as first shown by NMR studies on the alpha-galactosidase GalA from ''Thermotoga maritima'' <cite>Comfort2007</cite>. Correspondingly, GH36 enzymes use a classical [[Koshland double-displacement mechanism]] <cite>Sinnott1990</cite>, like their [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] relatives in Clan GH-D. This mechanism involves the formation of a covalent glycosyl-enzyme [[intermediate]] <cite>Vocadlo2001</cite> that partitions predominantly to water in hydrolytic enzymes and to saccharide acceptor substrates in transglycosylating enzymes, such as stachyose and raffinose synthases.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Family GH36 <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidases are anomeric configuration-[[retaining]] enzymes, as first shown by NMR studies on the <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase GalA from ''Thermotoga maritima'' <cite>Comfort2007</cite>. Correspondingly, GH36 enzymes use a classical [[Koshland double-displacement mechanism]] <cite>Sinnott1990</cite>, like their [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] relatives in Clan GH-D. This mechanism involves the formation of a covalent glycosyl-enzyme [[intermediate]] <cite>Vocadlo2001</cite> that partitions predominantly to water in hydrolytic enzymes and to saccharide acceptor substrates in transglycosylating enzymes, such as stachyose and raffinose synthases.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Catalytic Residues ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Catalytic Residues ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of archaeal GH36 alpha-galactosidases within [[Sequence-based classification of glycoside hydrolases|Clan]] GH-D originally highlighted likely candidates for the [[catalytic nucleophile]] and [[general acid/base]] residues in this family, based on protein sequence similarity with those identified in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] <cite>Brouns2006</cite>. Mutagenesis of the corresponding residues in ''Sulfolobus solfataricus'' alpha-galactosidase GalS dramatically reduced enzyme activity: the D367G ([[catalytic nucleophile]]) and D425G ([[general acid/base]]) mutant had <1 x 10<sup>–3</sup> and 5 x 10<sup>–3</sup> lower activity than the wild type enzyme when assayed against ''p''-nitrophenyl alpha-D-galactopyranoside <cite>Brouns2006</cite>. Rescue of the catalytic function of both enzyme mutants was unsuccessful with both azide and formate anions <cite>Brouns2006</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of archaeal GH36 <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidases within [[Sequence-based classification of glycoside hydrolases|Clan]] GH-D originally highlighted likely candidates for the [[catalytic nucleophile]] and [[general acid/base]] residues in this family, based on protein sequence similarity with those identified in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] <cite>Brouns2006</cite>. Mutagenesis of the corresponding residues in ''Sulfolobus solfataricus'' <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase GalS dramatically reduced enzyme activity: the D367G ([[catalytic nucleophile]]) and D425G ([[general acid/base]]) mutant had <1 x 10<sup>–3</sup> and 5 x 10<sup>–3</sup> lower activity than the wild type enzyme when assayed against ''p''-nitrophenyl <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-D-galactopyranoside <cite>Brouns2006</cite>. Rescue of the catalytic function of both enzyme mutants was unsuccessful with both azide and formate anions <cite>Brouns2006</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>The identities of the catalytic residues in GH36 were also confirmed in the ''Thermotoga maritima'' alpha-galactosidase GalA, guided by structural homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes <cite>Comfort2007</cite>. Site-directed mutation of Asp327 to Gly yielded a variant that had a 200-800-fold lower rate on aryl galactosides compared with the WT enzyme. Addition of azide was shown to rescue the ability of the enzyme to cleave ''p''-nitrophenyl alpha-D-galactopyranoside and resulted in formation of beta-galactopyranosyl azide, confirming Asp327 as the [[catalytic nucleophile]]. Mutation of the predicted [[general acid/base]] residue, Asp387, to Gly reduced activity 1500-fold on ''p''-nitrophenyl alpha-D-galactopyranoside, while addition of azide resulted in formation of alpha-galactopyranosyl azide by nucleophilic attack on the beta-linked glycosyl enzyme [[intermediate]].</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>The identities of the catalytic residues in GH36 were also confirmed in the ''Thermotoga maritima'' <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase GalA, guided by structural homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes <cite>Comfort2007</cite>. Site-directed mutation of Asp327 to Gly yielded a variant that had a 200-800-fold lower rate on aryl galactosides compared with the WT enzyme. Addition of azide was shown to rescue the ability of the enzyme to cleave ''p''-nitrophenyl <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-D-galactopyranoside and resulted in formation of <ins class="diffchange diffchange-inline">&</ins>beta<ins class="diffchange diffchange-inline">;</ins>-galactopyranosyl azide, confirming Asp327 as the [[catalytic nucleophile]]. Mutation of the predicted [[general acid/base]] residue, Asp387, to Gly reduced activity 1500-fold on ''p''-nitrophenyl <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-D-galactopyranoside, while addition of azide resulted in formation of <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactopyranosyl azide by nucleophilic attack on the <ins class="diffchange diffchange-inline">&</ins>beta<ins class="diffchange diffchange-inline">;</ins>-linked glycosyl enzyme [[intermediate]].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Three-dimensional structures ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Three-dimensional structures ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>In June 2005, the first three-dimensional structural coordinates for a member of this family, alpha-galactosidase TmGalA from ''Thermotoga maritima'', were deposited by the Joint Center For Structural Genomics (JCSG) (X-ray, 2.34 Å, [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1zy9 PDB 1zy9]) <cite>Lesley2002</cite>. Subsequent analysis of this data in the context of existing structures of [[GH27]] enzymes, revealed homologous active site residues, including two key catalytic Asp residues <cite>Comfort2007 Ernst2006</cite> and a number of conserved substrate-binding residues <cite>Comfort2007</cite>. The active sites of both [[GH27]] and GH36 enzymes are presented by (alpha/beta)8 (TIM) barrel domains. Both GH36 and [[GH27]] enzymes contain a C-terminal beta-sheet domain of unknown function, although this domain is structurally different and more disordered in TmGalA compared with the homologous domain in [[GH27]] (e.g., ''Oryza sativa'' alpha-galactosidase, [{{PDBlink}}1uas PDB 1uas]). Notably, an extra N-terminal, primarily beta-sheet domain, which is not found in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes, contributes a key substrate-binding residue (Trp65) to the active site of TmGalA (Trp65, replacing Trp164 in the ''O. sativa'' enzyme). Another notable active site substitution, this time within the TIM barrel itself, is the replacement of the aromatic residues Phe328 and Trp 291 in TmGalA with Ser102 and the Cys101-Cys132 disulfide in the ''O. sativa'' enzyme <cite>Comfort2007</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>In June 2005, the first three-dimensional structural coordinates for a member of this family, <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase TmGalA from ''Thermotoga maritima'', were deposited by the Joint Center For Structural Genomics (JCSG) (X-ray, 2.34 Å, [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1zy9 PDB 1zy9]) <cite>Lesley2002</cite>. Subsequent analysis of this data in the context of existing structures of [[GH27]] enzymes, revealed homologous active site residues, including two key catalytic Asp residues <cite>Comfort2007 Ernst2006</cite> and a number of conserved substrate-binding residues <cite>Comfort2007</cite>. The active sites of both [[GH27]] and GH36 enzymes are presented by (<ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>/<ins class="diffchange diffchange-inline">&</ins>beta<ins class="diffchange diffchange-inline">;</ins>)<ins class="diffchange diffchange-inline"><sub></ins>8<ins class="diffchange diffchange-inline"></sub> </ins>(TIM) barrel domains. Both GH36 and [[GH27]] enzymes contain a C-terminal <ins class="diffchange diffchange-inline">&</ins>beta<ins class="diffchange diffchange-inline">;</ins>-sheet domain of unknown function, although this domain is structurally different and more disordered in TmGalA compared with the homologous domain in [[GH27]] (e.g., ''Oryza sativa'' <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase, [{{PDBlink}}1uas PDB 1uas]). Notably, an extra N-terminal, primarily <ins class="diffchange diffchange-inline">&</ins>beta<ins class="diffchange diffchange-inline">;</ins>-sheet domain, which is not found in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes, contributes a key substrate-binding residue (Trp65) to the active site of TmGalA (Trp65, replacing Trp164 in the ''O. sativa'' enzyme). Another notable active site substitution, this time within the TIM barrel itself, is the replacement of the aromatic residues Phe328 and Trp 291 in TmGalA with Ser102 and the Cys101-Cys132 disulfide in the ''O. sativa'' enzyme <cite>Comfort2007</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of Clan GH-D, comprised of GH36 and [[Glycoside Hydrolase Family GH27 (GH27)|GH27]], has indicated that archaeal GH36 enzymes are more closely related to plant "alkaline" alpha-galactosidases, raffinose synthases, and stachyose synthases than to bacterial and fungal members. Thermophilic bacterial sequences, such as TmGalA, appear to form a divergent cluster within this latter subgroup <cite>Brouns2006</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of Clan GH-D, comprised of GH36 and [[Glycoside Hydrolase Family GH27 (GH27)|GH27]], has indicated that archaeal GH36 enzymes are more closely related to plant "alkaline" <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidases, raffinose synthases, and stachyose synthases than to bacterial and fungal members. Thermophilic bacterial sequences, such as TmGalA, appear to form a divergent cluster within this latter subgroup <cite>Brouns2006</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Family Firsts ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Family Firsts ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>;First sterochemistry determination: ''Thermotoga maritima'' alpha-galactosidase, by NMR <cite>Comfort2007</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>;First sterochemistry determination: ''Thermotoga maritima'' <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase, by NMR <cite>Comfort2007</cite>.</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>;First [[catalytic nucleophile]] identification: ''Sulfolobus solfataricus'' alpha-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite>Brouns2006</cite>. Subsequently confirmed in ''Thermotoga maritima'' alpha-galactosidase by structural homology, mutagenesis, and azide rescue <cite>Comfort2007</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>;First [[catalytic nucleophile]] identification: ''Sulfolobus solfataricus'' <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite>Brouns2006</cite>. Subsequently confirmed in ''Thermotoga maritima'' <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase by structural homology, mutagenesis, and azide rescue <cite>Comfort2007</cite>.</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>;First [[general acid/base]] residue identification: ''Sulfolobus solfataricus'' alpha-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite>Brouns2006</cite>. Subsequently confirmed in ''Thermotoga maritima'' alpha-galactosidase by structural homology, mutagenesis, and azide rescue <cite>Comfort2007</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>;First [[general acid/base]] residue identification: ''Sulfolobus solfataricus'' <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite>Brouns2006</cite>. Subsequently confirmed in ''Thermotoga maritima'' <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase by structural homology, mutagenesis, and azide rescue <cite>Comfort2007</cite>.</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>;First 3-D structure: ''Thermotoga maritima'' alpha-galactosidase by X-ray crystallography. Coordinates ([{{PDBlink}}1zy9 PDB 1zy9]) deposited in 2005 as part of a high-throughput functional genomics project <cite>Lesley2002</cite>, structural analyses published by other groups in 2006 and 2007 <cite>Comfort2007 Ernst2006</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>;First 3-D structure: ''Thermotoga maritima'' <ins class="diffchange diffchange-inline">&</ins>alpha<ins class="diffchange diffchange-inline">;</ins>-galactosidase by X-ray crystallography. Coordinates ([{{PDBlink}}1zy9 PDB 1zy9]) deposited in 2005 as part of a high-throughput functional genomics project <cite>Lesley2002</cite>, structural analyses published by other groups in 2006 and 2007 <cite>Comfort2007 Ernst2006</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== References ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== References ==</div></td></tr>
</table>
Spencer Williams
https://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_36&diff=5255&oldid=prev
Harry Brumer: Changing reference indices to AuthorYear format
2010-07-30T06:27:30Z
<p>Changing reference indices to AuthorYear format</p>
<table class="diff diff-contentalign-left diff-editfont-monospace" data-mw="interface">
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 06:27, 30 July 2010</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Kinetics and Mechanism ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Kinetics and Mechanism ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Family GH36 alpha-galactosidases are anomeric configuration-[[retaining]] enzymes, as first shown by NMR studies on the alpha-galactosidase GalA from ''Thermotoga maritima'' <cite><del class="diffchange diffchange-inline">1</del></cite>. Correspondingly, GH36 enzymes use a classical [[Koshland double-displacement mechanism]] <cite><del class="diffchange diffchange-inline">4</del></cite>, like their [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] relatives in Clan GH-D. This mechanism involves the formation of a covalent glycosyl-enzyme [[intermediate]] <cite><del class="diffchange diffchange-inline">5</del></cite> that partitions predominantly to water in hydrolytic enzymes and to saccharide acceptor substrates in transglycosylating enzymes, such as stachyose and raffinose synthases.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Family GH36 alpha-galactosidases are anomeric configuration-[[retaining]] enzymes, as first shown by NMR studies on the alpha-galactosidase GalA from ''Thermotoga maritima'' <cite><ins class="diffchange diffchange-inline">Comfort2007</ins></cite>. Correspondingly, GH36 enzymes use a classical [[Koshland double-displacement mechanism]] <cite><ins class="diffchange diffchange-inline">Sinnott1990</ins></cite>, like their [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] relatives in Clan GH-D. This mechanism involves the formation of a covalent glycosyl-enzyme [[intermediate]] <cite><ins class="diffchange diffchange-inline">Vocadlo2001</ins></cite> that partitions predominantly to water in hydrolytic enzymes and to saccharide acceptor substrates in transglycosylating enzymes, such as stachyose and raffinose synthases.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Catalytic Residues ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Catalytic Residues ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of archaeal GH36 alpha-galactosidases within [[Sequence-based classification of glycoside hydrolases|Clan]] GH-D originally highlighted likely candidates for the [[catalytic nucleophile]] and [[general acid/base]] residues in this family, based on protein sequence similarity with those identified in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] <cite><del class="diffchange diffchange-inline">3</del></cite>. Mutagenesis of the corresponding residues in ''Sulfolobus solfataricus'' alpha-galactosidase GalS dramatically reduced enzyme activity: the D367G ([[catalytic nucleophile]]) and D425G ([[general acid/base]]) mutant had <1 x 10<sup>–3</sup> and 5 x 10<sup>–3</sup> lower activity than the wild type enzyme when assayed against ''p''-nitrophenyl alpha-D-galactopyranoside <cite><del class="diffchange diffchange-inline">3</del></cite>. Rescue of the catalytic function of both enzyme mutants was unsuccessful with both azide and formate anions <cite><del class="diffchange diffchange-inline">3</del></cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of archaeal GH36 alpha-galactosidases within [[Sequence-based classification of glycoside hydrolases|Clan]] GH-D originally highlighted likely candidates for the [[catalytic nucleophile]] and [[general acid/base]] residues in this family, based on protein sequence similarity with those identified in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] <cite><ins class="diffchange diffchange-inline">Brouns2006</ins></cite>. Mutagenesis of the corresponding residues in ''Sulfolobus solfataricus'' alpha-galactosidase GalS dramatically reduced enzyme activity: the D367G ([[catalytic nucleophile]]) and D425G ([[general acid/base]]) mutant had <1 x 10<sup>–3</sup> and 5 x 10<sup>–3</sup> lower activity than the wild type enzyme when assayed against ''p''-nitrophenyl alpha-D-galactopyranoside <cite><ins class="diffchange diffchange-inline">Brouns2006</ins></cite>. Rescue of the catalytic function of both enzyme mutants was unsuccessful with both azide and formate anions <cite><ins class="diffchange diffchange-inline">Brouns2006</ins></cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>The identities of the catalytic residues in GH36 were also confirmed in the ''Thermotoga maritima'' alpha-galactosidase GalA, guided by structural homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes <cite><del class="diffchange diffchange-inline">1</del></cite>. Site-directed mutation of Asp327 to Gly yielded a variant that had a 200-800-fold lower rate on aryl galactosides compared with the WT enzyme. Addition of azide was shown to rescue the ability of the enzyme to cleave ''p''-nitrophenyl alpha-D-galactopyranoside and resulted in formation of beta-galactopyranosyl azide, confirming Asp327 as the [[catalytic nucleophile]]. Mutation of the predicted [[general acid/base]] residue, Asp387, to Gly reduced activity 1500-fold on ''p''-nitrophenyl alpha-D-galactopyranoside, while addition of azide resulted in formation of alpha-galactopyranosyl azide by nucleophilic attack on the beta-linked glycosyl enzyme [[intermediate]].</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>The identities of the catalytic residues in GH36 were also confirmed in the ''Thermotoga maritima'' alpha-galactosidase GalA, guided by structural homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes <cite><ins class="diffchange diffchange-inline">Comfort2007</ins></cite>. Site-directed mutation of Asp327 to Gly yielded a variant that had a 200-800-fold lower rate on aryl galactosides compared with the WT enzyme. Addition of azide was shown to rescue the ability of the enzyme to cleave ''p''-nitrophenyl alpha-D-galactopyranoside and resulted in formation of beta-galactopyranosyl azide, confirming Asp327 as the [[catalytic nucleophile]]. Mutation of the predicted [[general acid/base]] residue, Asp387, to Gly reduced activity 1500-fold on ''p''-nitrophenyl alpha-D-galactopyranoside, while addition of azide resulted in formation of alpha-galactopyranosyl azide by nucleophilic attack on the beta-linked glycosyl enzyme [[intermediate]].</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Three-dimensional structures ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Three-dimensional structures ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>In June 2005, the first three-dimensional structural coordinates for a member of this family, alpha-galactosidase TmGalA from ''Thermotoga maritima'', were deposited by the Joint Center For Structural Genomics (JCSG) (X-ray, 2.34 Å, [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1zy9 PDB 1zy9]) <cite><del class="diffchange diffchange-inline">2</del></cite>. Subsequent analysis of this data in the context of existing structures of [[GH27]] enzymes, revealed homologous active site residues, including two key catalytic Asp residues <cite><del class="diffchange diffchange-inline">1 6</del></cite> and a number of conserved substrate-binding residues <cite><del class="diffchange diffchange-inline">1</del></cite>. The active sites of both [[GH27]] and GH36 enzymes are presented by (alpha/beta)8 (TIM) barrel domains. Both GH36 and [[GH27]] enzymes contain a C-terminal beta-sheet domain of unknown function, although this domain is structurally different and more disordered in TmGalA compared with the homologous domain in [[GH27]] (e.g., ''Oryza sativa'' alpha-galactosidase, [{{PDBlink}}1uas PDB 1uas]). Notably, an extra N-terminal, primarily beta-sheet domain, which is not found in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes, contributes a key substrate-binding residue (Trp65) to the active site of TmGalA (Trp65, replacing Trp164 in the ''O. sativa'' enzyme). Another notable active site substitution, this time within the TIM barrel itself, is the replacement of the aromatic residues Phe328 and Trp 291 in TmGalA with Ser102 and the Cys101-Cys132 disulfide in the ''O. sativa'' enzyme <cite><del class="diffchange diffchange-inline">1</del></cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>In June 2005, the first three-dimensional structural coordinates for a member of this family, alpha-galactosidase TmGalA from ''Thermotoga maritima'', were deposited by the Joint Center For Structural Genomics (JCSG) (X-ray, 2.34 Å, [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1zy9 PDB 1zy9]) <cite><ins class="diffchange diffchange-inline">Lesley2002</ins></cite>. Subsequent analysis of this data in the context of existing structures of [[GH27]] enzymes, revealed homologous active site residues, including two key catalytic Asp residues <cite><ins class="diffchange diffchange-inline">Comfort2007 Ernst2006</ins></cite> and a number of conserved substrate-binding residues <cite><ins class="diffchange diffchange-inline">Comfort2007</ins></cite>. The active sites of both [[GH27]] and GH36 enzymes are presented by (alpha/beta)8 (TIM) barrel domains. Both GH36 and [[GH27]] enzymes contain a C-terminal beta-sheet domain of unknown function, although this domain is structurally different and more disordered in TmGalA compared with the homologous domain in [[GH27]] (e.g., ''Oryza sativa'' alpha-galactosidase, [{{PDBlink}}1uas PDB 1uas]). Notably, an extra N-terminal, primarily beta-sheet domain, which is not found in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes, contributes a key substrate-binding residue (Trp65) to the active site of TmGalA (Trp65, replacing Trp164 in the ''O. sativa'' enzyme). Another notable active site substitution, this time within the TIM barrel itself, is the replacement of the aromatic residues Phe328 and Trp 291 in TmGalA with Ser102 and the Cys101-Cys132 disulfide in the ''O. sativa'' enzyme <cite><ins class="diffchange diffchange-inline">Comfort2007</ins></cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of Clan GH-D, comprised of GH36 and [[Glycoside Hydrolase Family GH27 (GH27)|GH27]], has indicated that archaeal GH36 enzymes are more closely related to plant "alkaline" alpha-galactosidases, raffinose synthases, and stachyose synthases than to bacterial and fungal members. Thermophilic bacterial sequences, such as TmGalA, appear to form a divergent cluster within this latter subgroup <cite><del class="diffchange diffchange-inline">3</del></cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of Clan GH-D, comprised of GH36 and [[Glycoside Hydrolase Family GH27 (GH27)|GH27]], has indicated that archaeal GH36 enzymes are more closely related to plant "alkaline" alpha-galactosidases, raffinose synthases, and stachyose synthases than to bacterial and fungal members. Thermophilic bacterial sequences, such as TmGalA, appear to form a divergent cluster within this latter subgroup <cite><ins class="diffchange diffchange-inline">Brouns2006</ins></cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Family Firsts ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Family Firsts ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>;First sterochemistry determination: ''Thermotoga maritima'' alpha-galactosidase, by NMR <cite><del class="diffchange diffchange-inline">1</del></cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>;First sterochemistry determination: ''Thermotoga maritima'' alpha-galactosidase, by NMR <cite><ins class="diffchange diffchange-inline">Comfort2007</ins></cite>.</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>;First [[catalytic nucleophile]] identification: ''Sulfolobus solfataricus'' alpha-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite><del class="diffchange diffchange-inline">3</del></cite>. Subsequently confirmed in ''Thermotoga maritima'' alpha-galactosidase by structural homology, mutagenesis, and azide rescue <cite><del class="diffchange diffchange-inline">1</del></cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>;First [[catalytic nucleophile]] identification: ''Sulfolobus solfataricus'' alpha-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite><ins class="diffchange diffchange-inline">Brouns2006</ins></cite>. Subsequently confirmed in ''Thermotoga maritima'' alpha-galactosidase by structural homology, mutagenesis, and azide rescue <cite><ins class="diffchange diffchange-inline">Comfort2007</ins></cite>.</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>;First [[general acid/base]] residue identification: ''Sulfolobus solfataricus'' alpha-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite><del class="diffchange diffchange-inline">3</del></cite>. Subsequently confirmed in ''Thermotoga maritima'' alpha-galactosidase by structural homology, mutagenesis, and azide rescue <cite><del class="diffchange diffchange-inline">1</del></cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>;First [[general acid/base]] residue identification: ''Sulfolobus solfataricus'' alpha-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite><ins class="diffchange diffchange-inline">Brouns2006</ins></cite>. Subsequently confirmed in ''Thermotoga maritima'' alpha-galactosidase by structural homology, mutagenesis, and azide rescue <cite><ins class="diffchange diffchange-inline">Comfort2007</ins></cite>.</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>;First 3-D structure: ''Thermotoga maritima'' alpha-galactosidase by X-ray crystallography. Coordinates ([{{PDBlink}}1zy9 PDB 1zy9]) deposited in 2005 as part of a high-throughput functional genomics project <cite><del class="diffchange diffchange-inline">2</del></cite>, structural analyses published by other groups in 2006 and 2007 <cite><del class="diffchange diffchange-inline">1 6</del></cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>;First 3-D structure: ''Thermotoga maritima'' alpha-galactosidase by X-ray crystallography. Coordinates ([{{PDBlink}}1zy9 PDB 1zy9]) deposited in 2005 as part of a high-throughput functional genomics project <cite><ins class="diffchange diffchange-inline">Lesley2002</ins></cite>, structural analyses published by other groups in 2006 and 2007 <cite><ins class="diffchange diffchange-inline">Comfort2007 Ernst2006</ins></cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== References ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== References ==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><biblio></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div><biblio></div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>#<del class="diffchange diffchange-inline">1 </del>pmid=17323919</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>#<ins class="diffchange diffchange-inline">Comfort2007 </ins>pmid=17323919</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>#<del class="diffchange diffchange-inline">2 </del>pmid=12193646</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>#<ins class="diffchange diffchange-inline">Lesley2002 </ins>pmid=12193646</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>#<del class="diffchange diffchange-inline">3 </del>pmid=16547025</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>#<ins class="diffchange diffchange-inline">Brouns2006 </ins>pmid=16547025</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>#<del class="diffchange diffchange-inline">4 </del>Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. [http://dx.doi.org/10.1021/cr00105a006 DOI: 10.1021/cr00105a006]</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>#<ins class="diffchange diffchange-inline">Sinnott1990 </ins>Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. [http://dx.doi.org/10.1021/cr00105a006 DOI: 10.1021/cr00105a006]</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>#<del class="diffchange diffchange-inline">5 </del>pmid=11518970</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>#<ins class="diffchange diffchange-inline">Vocadlo2001 </ins>pmid=11518970</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>#<del class="diffchange diffchange-inline">6 </del>pmid=16580018</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>#<ins class="diffchange diffchange-inline">Ernst2006 </ins>pmid=16580018</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div> </div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div> </div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div> </div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div></biblio></div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div></biblio></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>[[Category:Glycoside Hydrolase Families|GH036]]</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>[[Category:Glycoside Hydrolase Families|GH036]]</div></td></tr>
</table>
Harry Brumer
https://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_36&diff=5027&oldid=prev
Spencer Williams at 11:00, 22 June 2010
2010-06-22T11:00:23Z
<p></p>
<table class="diff diff-contentalign-left diff-editfont-monospace" data-mw="interface">
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 11:00, 22 June 2010</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Catalytic Residues ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Catalytic Residues ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of archaeal GH36 alpha-galactosidases within Clan GH-D originally highlighted likely candidates for the [[catalytic nucleophile]] and [[general acid/base]] residues in this family, based on protein sequence similarity with those identified in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] <cite>3</cite>. Mutagenesis of the corresponding residues in ''Sulfolobus solfataricus'' alpha-galactosidase GalS dramatically reduced enzyme activity: the D367G ([[catalytic nucleophile]]) and D425G ([[general acid/base]]) mutant had <1 x 10<sup>–3</sup> and 5 x 10<sup>–3</sup> lower activity than the wild type enzyme when assayed against ''p''-nitrophenyl alpha-D-galactopyranoside <cite>3</cite>. Rescue of the catalytic function of both enzyme mutants was unsuccessful with both azide and formate anions <cite>3</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of archaeal GH36 alpha-galactosidases within <ins class="diffchange diffchange-inline">[[Sequence-based classification of glycoside hydrolases|</ins>Clan<ins class="diffchange diffchange-inline">]] </ins>GH-D originally highlighted likely candidates for the [[catalytic nucleophile]] and [[general acid/base]] residues in this family, based on protein sequence similarity with those identified in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] <cite>3</cite>. Mutagenesis of the corresponding residues in ''Sulfolobus solfataricus'' alpha-galactosidase GalS dramatically reduced enzyme activity: the D367G ([[catalytic nucleophile]]) and D425G ([[general acid/base]]) mutant had <1 x 10<sup>–3</sup> and 5 x 10<sup>–3</sup> lower activity than the wild type enzyme when assayed against ''p''-nitrophenyl alpha-D-galactopyranoside <cite>3</cite>. Rescue of the catalytic function of both enzyme mutants was unsuccessful with both azide and formate anions <cite>3</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>The identities of the catalytic residues in GH36 were also confirmed in the ''Thermotoga maritima'' alpha-galactosidase GalA, guided by structural homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes <cite>1</cite>. Site-directed mutation of Asp327 to Gly yielded a variant that had a 200-800-fold lower rate on aryl galactosides compared with the WT enzyme. Addition of azide was shown to rescue the ability of the enzyme to cleave ''p''-nitrophenyl alpha-D-galactopyranoside and resulted in formation of beta-galactopyranosyl azide, confirming Asp327 as the [[catalytic nucleophile]]. Mutation of the predicted [[general acid/base]] residue, Asp387, to Gly reduced activity 1500-fold on ''p''-nitrophenyl alpha-D-galactopyranoside, while addition of azide resulted in formation of alpha-galactopyranosyl azide by nucleophilic attack on the beta-linked glycosyl enzyme [[intermediate]].</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>The identities of the catalytic residues in GH36 were also confirmed in the ''Thermotoga maritima'' alpha-galactosidase GalA, guided by structural homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes <cite>1</cite>. Site-directed mutation of Asp327 to Gly yielded a variant that had a 200-800-fold lower rate on aryl galactosides compared with the WT enzyme. Addition of azide was shown to rescue the ability of the enzyme to cleave ''p''-nitrophenyl alpha-D-galactopyranoside and resulted in formation of beta-galactopyranosyl azide, confirming Asp327 as the [[catalytic nucleophile]]. Mutation of the predicted [[general acid/base]] residue, Asp387, to Gly reduced activity 1500-fold on ''p''-nitrophenyl alpha-D-galactopyranoside, while addition of azide resulted in formation of alpha-galactopyranosyl azide by nucleophilic attack on the beta-linked glycosyl enzyme [[intermediate]].</div></td></tr>
</table>
Spencer Williams
https://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_36&diff=3457&oldid=prev
Harry Brumer: /* Family Firsts */ updated with PDBlink template
2010-01-14T16:49:19Z
<p><span dir="auto"><span class="autocomment">Family Firsts: </span> updated with PDBlink template</span></p>
<table class="diff diff-contentalign-left diff-editfont-monospace" data-mw="interface">
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<col class="diff-content" />
<col class="diff-marker" />
<col class="diff-content" />
<tr class="diff-title" lang="en-CA">
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 16:49, 14 January 2010</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l45" >Line 45:</td>
<td colspan="2" class="diff-lineno">Line 45:</td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>;First [[catalytic nucleophile]] identification: ''Sulfolobus solfataricus'' alpha-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite>3</cite>. Subsequently confirmed in ''Thermotoga maritima'' alpha-galactosidase by structural homology, mutagenesis, and azide rescue <cite>1</cite>.</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>;First [[catalytic nucleophile]] identification: ''Sulfolobus solfataricus'' alpha-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite>3</cite>. Subsequently confirmed in ''Thermotoga maritima'' alpha-galactosidase by structural homology, mutagenesis, and azide rescue <cite>1</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>;First [[general acid/base]] residue identification: ''Sulfolobus solfataricus'' alpha-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite>3</cite>. Subsequently confirmed in ''Thermotoga maritima'' alpha-galactosidase by structural homology, mutagenesis, and azide rescue <cite>1</cite>.</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>;First [[general acid/base]] residue identification: ''Sulfolobus solfataricus'' alpha-galactosidase GalS, by sequence homology with [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes and mutagenesis <cite>3</cite>. Subsequently confirmed in ''Thermotoga maritima'' alpha-galactosidase by structural homology, mutagenesis, and azide rescue <cite>1</cite>.</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>;First 3-D structure: ''Thermotoga maritima'' alpha-galactosidase by X-ray crystallography. Coordinates ([<del class="diffchange diffchange-inline">http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=</del>1zy9 PDB 1zy9]) deposited in 2005 as part of a high-throughput functional genomics project <cite>2</cite>, structural analyses published by other groups in 2006 and 2007 <cite>1 6</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>;First 3-D structure: ''Thermotoga maritima'' alpha-galactosidase by X-ray crystallography. Coordinates ([<ins class="diffchange diffchange-inline">{{PDBlink}}</ins>1zy9 PDB 1zy9]) deposited in 2005 as part of a high-throughput functional genomics project <cite>2</cite>, structural analyses published by other groups in 2006 and 2007 <cite>1 6</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== References ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== References ==</div></td></tr>
</table>
Harry Brumer
https://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_36&diff=3456&oldid=prev
Harry Brumer: /* Three-dimensional structures */ updated with PDBlink template
2010-01-14T16:48:50Z
<p><span dir="auto"><span class="autocomment">Three-dimensional structures: </span> updated with PDBlink template</span></p>
<table class="diff diff-contentalign-left diff-editfont-monospace" data-mw="interface">
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 16:48, 14 January 2010</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l37" >Line 37:</td>
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<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Three-dimensional structures ==</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Three-dimensional structures ==</div></td></tr>
<tr><td class='diff-marker'>−</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>In June 2005, the first three-dimensional structural coordinates for a member of this family, alpha-galactosidase TmGalA from ''Thermotoga maritima'', were deposited by the Joint Center For Structural Genomics (JCSG) (X-ray, 2.34 Å, [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1zy9 PDB 1zy9]) <cite>2</cite>. Subsequent analysis of this data in the context of existing structures of [[GH27]] enzymes, revealed homologous active site residues, including two key catalytic Asp residues <cite>1 6</cite> and a number of conserved substrate-binding residues <cite>1</cite>. The active sites of both [[GH27]] and GH36 enzymes are presented by (alpha/beta)8 (TIM) barrel domains. Both GH36 and [[GH27]] enzymes contain a C-terminal beta-sheet domain of unknown function, although this domain is structurally different and more disordered in TmGalA compared with the homologous domain in [[GH27]] (e.g., ''Oryza sativa'' alpha-galactosidase, [<del class="diffchange diffchange-inline">http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=</del>1uas PDB 1uas]). Notably, an extra N-terminal, primarily beta-sheet domain, which is not found in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes, contributes a key substrate-binding residue (Trp65) to the active site of TmGalA (Trp65, replacing Trp164 in the ''O. sativa'' enzyme). Another notable active site substitution, this time within the TIM barrel itself, is the replacement of the aromatic residues Phe328 and Trp 291 in TmGalA with Ser102 and the Cys101-Cys132 disulfide in the ''O. sativa'' enzyme <cite>1</cite>.</div></td><td class='diff-marker'>+</td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>In June 2005, the first three-dimensional structural coordinates for a member of this family, alpha-galactosidase TmGalA from ''Thermotoga maritima'', were deposited by the Joint Center For Structural Genomics (JCSG) (X-ray, 2.34 Å, [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1zy9 PDB 1zy9]) <cite>2</cite>. Subsequent analysis of this data in the context of existing structures of [[GH27]] enzymes, revealed homologous active site residues, including two key catalytic Asp residues <cite>1 6</cite> and a number of conserved substrate-binding residues <cite>1</cite>. The active sites of both [[GH27]] and GH36 enzymes are presented by (alpha/beta)8 (TIM) barrel domains. Both GH36 and [[GH27]] enzymes contain a C-terminal beta-sheet domain of unknown function, although this domain is structurally different and more disordered in TmGalA compared with the homologous domain in [[GH27]] (e.g., ''Oryza sativa'' alpha-galactosidase, [<ins class="diffchange diffchange-inline">{{PDBlink}}</ins>1uas PDB 1uas]). Notably, an extra N-terminal, primarily beta-sheet domain, which is not found in [[Glycoside Hydrolase Family GH27 (GH27)|GH27]] enzymes, contributes a key substrate-binding residue (Trp65) to the active site of TmGalA (Trp65, replacing Trp164 in the ''O. sativa'' enzyme). Another notable active site substitution, this time within the TIM barrel itself, is the replacement of the aromatic residues Phe328 and Trp 291 in TmGalA with Ser102 and the Cys101-Cys132 disulfide in the ''O. sativa'' enzyme <cite>1</cite>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of Clan GH-D, comprised of GH36 and [[Glycoside Hydrolase Family GH27 (GH27)|GH27]], has indicated that archaeal GH36 enzymes are more closely related to plant "alkaline" alpha-galactosidases, raffinose synthases, and stachyose synthases than to bacterial and fungal members. Thermophilic bacterial sequences, such as TmGalA, appear to form a divergent cluster within this latter subgroup <cite>3</cite>.</div></td><td class='diff-marker'> </td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>Detailed phylogenetic analysis of Clan GH-D, comprised of GH36 and [[Glycoside Hydrolase Family GH27 (GH27)|GH27]], has indicated that archaeal GH36 enzymes are more closely related to plant "alkaline" alpha-galactosidases, raffinose synthases, and stachyose synthases than to bacterial and fungal members. Thermophilic bacterial sequences, such as TmGalA, appear to form a divergent cluster within this latter subgroup <cite>3</cite>.</div></td></tr>
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Harry Brumer
https://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_36&diff=2676&oldid=prev
Harry Brumer at 17:06, 3 November 2009
2009-11-03T17:06:32Z
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Harry Brumer