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| | |'''Clan''' | | |'''Clan''' |
| − | |not assigned | + | |GH-x |
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| | |'''Mechanism''' | | |'''Mechanism''' |
| − | |retaining | + | |retaining/inverting |
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| | |'''Active site residues''' | | |'''Active site residues''' |
| − | |known | + | |known/not known |
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| | |{{Hl2}} colspan="2" align="center" |'''CAZy DB link''' | | |{{Hl2}} colspan="2" align="center" |'''CAZy DB link''' |
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| | == Substrate specificities == | | == Substrate specificities == |
| − | [[Glycoside hydrolases]] of family GH99 are [[endo]]-acting α-mannosidases that cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc<sub>1-3</sub>Man<sub>9</sub>GlcNAc<sub>2</sub>, and possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. This family was originally created from the mammalian enzyme, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Mammalian GH99 enzymes are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. Substrate studies on a bacterial GH99 enzyme from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, and the ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' enzymes <cite>Thompson2012</cite> have shown that these enzymes can also process Glc<sub>1/3</sub>Man<sub>9/7</sub>GlcNAc<sub>2</sub> structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc<sub>3</sub>Man<sub>9</sub>GlcNAc<sub>2</sub> structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.
| + | Content is to be added here. In the meantime, please see these references for an essential introduction to the CAZy classification system: <cite>DaviesSinnott2008 Cantarel2009</cite>. |
| | | | |
| | == Kinetics and Mechanism == | | == Kinetics and Mechanism == |
| − | Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by <sup>1</sup>H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.
| + | Content is to be added here. |
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| | == Catalytic Residues == | | == Catalytic Residues == |
| − | | + | Content is to be added here. |
| − | The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and zero activity against the natural substrate, Glc<sub>3</sub>Man<sub>7</sub>GlcNAc<sub>2</sub>, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the identity of the nucleophilic residue used for catalysis is more obscure and the 2OH of the substrate has been proposed to act as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012</cite>. According to this proposal, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.
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| | | | |
| | == Three-dimensional structures == | | == Three-dimensional structures == |
| − | Three-dimensional structures are available for bacterial members of GH99, including ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)<sub>8</sub> TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, the precise identity of the nucleophilic residue is unclear, as in all GH99-inhibitor complexes with an occupied -1 subsite there is no candidate nucleophile within a reasonable distance to the "anomeric" carbon: in ''Bx''GH99 Glu333 is approximately 3.5 Å distant and the OH of Tyr46 and Tyr252 are 4.0 Å distant.
| + | Content is to be added here. |
| − | | |
| − | === Sample structures ===
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| − | | |
| − | {| {{Prettytable}}
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| − | !style="width:50%"|Three-dimensional structure of GH99 endo-α-mannosidase from ''Bacteroides xylanisolvens'', PDB code [{{PDBlink}}4ad1] <cite>Thompson2012</cite>.
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| − | !style="width:50%"|Three-dimensional structure of GH99 endo-α-mannosidase from ''Bacteroides xylanisolvens'' bound to glucose-1,3-isofagomine and α-1,2- mannobiose, PDB code [{{PDBlink}}4ad4] <cite>Thompson2012</cite>.
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| − | <uploadedFileContents>4ad1.pdb</uploadedFileContents>
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| − | <script>cpk off; wireframe off; cartoon; color cartoon powderblue; select ligand; wireframe 0.3; select MG; spacefill; set spin Y 10; spin off; set antialiasDisplay OFF</script>
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| − | <frame>true</frame>
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| − | <uploadedFileContents>4ad4.pdb</uploadedFileContents>
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| − | <script>cpk off; wireframe off; cartoon; color cartoon powderblue; select ligand; wireframe 0.3; select MG; spacefill; set spin Y 10; spin off; set antialiasDisplay OFF</script>
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| − | </jmolApplet>
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| − | |}
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| − | <br style="clear: both" />
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| | | | |
| | == Family Firsts == | | == Family Firsts == |
| − | ;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by <sup>1</sup>H NMR <cite>Thompson2012</cite> | + | ;First stereochemistry determination: Content is to be added here. |
| − | | + | ;First catalytic nucleophile identification: Content is to be added here. |
| − | ;First [[catalytic nucleophile]] identification: It has been proposed that GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012</cite> | + | ;First general acid/base residue identification: Content is to be added here. |
| − | | + | ;First 3-D structure: Content is to be added here. |
| − | ;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite> | |
| − | | |
| − | ;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite> | |
| | | | |
| | == References == | | == References == |
| | <biblio> | | <biblio> |
| − | #Roth2003 pmid=12770767 | + | #Cantarel2009 pmid=18838391 |
| − | #Spiro1997 pmid=9361017 | + | #DaviesSinnott2008 Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. Biochem. J. (BJ Classic Paper, online only). [http://dx.doi.org/10.1042/BJ20080382 DOI: 10.1042/BJ20080382] |
| − | #Zuber2000 pmid=11102520
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| − | #Dale1986 pmid=3087421
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| − | #Kukushkin2011 pmid=21585340
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| − | #Spiro2000 pmid=10764841
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| − | #Thompson2012 pmid=22219371
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| − | #Matsuda2011 Matsuda K, Kurakata Y, Miyazaki T, Matsuo I, Ito Y, Nishikawa A, Tonozuka T. ''Heterologous expression, purification, and characterization of an α-mannosidase belonging to glycoside hydrolase family 99 of Shewanella amazonensis.'' Biosci Biotechnol Biochem. 2011;75(4):797-9. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1271/bbb.100874 DOI: 10.1271/bbb.100874]
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| − | [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=21512220 PMID:21512220]
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| − | #Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
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| | </biblio> | | </biblio> |
| | | | |
| | [[Category:Glycoside Hydrolase Families|GH099]] | | [[Category:Glycoside Hydrolase Families|GH099]] |
