Glycoside Hydrolase Family 99

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• Author: ^^^Spencer Williams^^^
• Responsible Curator: ^^^Gideon Davies^^^

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Glycoside Hydrolase Family GH99
Clan	not assigned
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH99.html

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_1-3Man₉GlcNAc₂, with maximal activity on the monoglucosylated forms [1]. This family was originally created from mammalian enzyme, cloned by Spiro and co-workers [2]. Mammalian GH99 enzymes are localized to the Golgi apparatus [3] 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 [4]. Mammalian endo-α-mannosidases has increased activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches [2]. There is evidence that mammalian endo-α-mannosidases act on dolichol-bound N-glycan precursors [5], as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway [6]. Substrate studies on a bacterial GH99 enzyme from Shewanella amazonensis [7], and the Bacteroides thetaiotaomicron Bacteroides xylanisolvens enzymes [8] have shown that these enzymes can also process Glc_1/3Man_9/7GlcNAc₂ structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of Bacteroides thetaiotaomicron on a fluorescently-labelled Glc₃Man₉GlcNAc₂ structure yielded kinetic parameters that were similar to that found for the mammalian enzyme [8]. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside [9] or α-glucosyl-1,3-α-mannopyranosyl fluoride [8], but are inactive on aryl mannosides.

Kinetics and Mechanism

Family GH99 endo-α-mannosidases are retaining enzymes, as first shown by ¹NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by Bacteroides thetaiotaomicron [8]. Retaining mannoside hydrolases (eg GH38) typically follow a classical Koshland double-displacement mechanism. However, X-ray crystallographic analysis of BxGH99 and BtGH99 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 [8]. In this proposal, Glu333 in BxGH99 (Glu329 in BtGH99) acts as a general acid/base to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

Catalytic Residues

The general acid/base was highlighted by X-ray crystallographic analysis as Glu336 in BxGH99 and Glu332 in BtGH99) [8]. The Glu332Ala mutant of BtGH99 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, Glc₃Man₇GlcNAc₂, 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[8]. According to this proposal, Glu333 in BxGH99 (Glu329 in BtGH99) acts as a general acid/base to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

Three-dimensional structures

Three-dimensional structures are available for bacterial members of GH99, including Bacteroides thetaiotaomicron and Bacteroides xylanisolvens[8]. They have a classical (β/α)₈ 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 'actice-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 BxGH99; Glu332 in BtGH99) 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 [8]. 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 BxGH99 Glu333 is approximately 3.5 Å distant and the OH of Tyr46 and Tyr252 are 4.0 Å distant.

Sample structures

Three-dimensional structure of GH99 endo-α-mannosidase from Bacteroides xylanisolvens, PDB code [1] [8].	Three-dimensional structure of GH99 endo-α-mannosidase from Bacteroides xylanisolvens bound to glucose-1,3-isofagomine and α-1,2- mannobiose, PDB code [2] [8].
<jmol> <jmolApplet> <color>white</color> <frame>true</frame> <uploadedFileContents>4ad1.pdb</uploadedFileContents> <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> </jmolApplet> </jmol>	<jmol> <jmolApplet> <color>white</color> <frame>true</frame> <uploadedFileContents>4ad4.pdb</uploadedFileContents> <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> </jmolApplet> </jmol>

Family Firsts

First sterochemistry determination

Bacteroides thetaiotaomicron endo-α-mannosidase by NMR [8]

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 [8]

First general acid/base residue identification

Bacteroides thetaiotaomicron endo-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities [8]

First 3-D structure of a GH99 enzyme

Bacteroides thetaiotaomicron and Bacteroides xylanisolvens endo-α-mannosidases [8]

References

1. Roth J, Ziak M, and Zuber C. (2003). The role of glucosidase II and endomannosidase in glucose trimming of asparagine-linked oligosaccharides. Biochimie. 2003;85(3-4):287-94. DOI:10.1016/s0300-9084(03)00049-x |
2. Spiro MJ, Bhoyroo VD, and Spiro RG. (1997). Molecular cloning and expression of rat liver endo-alpha-mannosidase, an N-linked oligosaccharide processing enzyme. J Biol Chem. 1997;272(46):29356-63. DOI:10.1074/jbc.272.46.29356 |
3. Zuber C, Spiro MJ, Guhl B, Spiro RG, and Roth J. (2000). Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control. Mol Biol Cell. 2000;11(12):4227-40. DOI:10.1091/mbc.11.12.4227 |
4. Dale MP, Kopfler WP, Chait I, and Byers LD. (1986). Beta-glucosidase: substrate, solvent, and viscosity variation as probes of the rate-limiting steps. Biochemistry. 1986;25(9):2522-9. DOI:10.1021/bi00357a036 |
5. Spiro MJ and Spiro RG. (2000). Use of recombinant endomannosidase for evaluation of the processing of N-linked oligosaccharides of glycoproteins and their oligosaccharide-lipid precursors. Glycobiology. 2000;10(5):521-9. DOI:10.1093/glycob/10.5.521 |
6. Kukushkin NV, Alonzi DS, Dwek RA, and Butters TD. (2011). Demonstration that endoplasmic reticulum-associated degradation of glycoproteins can occur downstream of processing by endomannosidase. Biochem J. 2011;438(1):133-42. DOI:10.1042/BJ20110186 |
7. Matsuda K, Kurakata Y, Miyazaki T, Matsuo I, Ito Y, Nishikawa A, and Tonozuka T. (2011). 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. DOI:10.1271/bbb.100874 |

8. http://www.pnas.org/content/early/2012/01/04/1111482109.abstract

9. Vogel, C.; Pohlentz, G. J. Carbohydr. Chem. 2000, 19, 1247-1258.