Glycoside Hydrolase Family 63

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• Author: Takashi Tonozuka and Takatsugu Miyazaki
• Responsible Curator: Takashi Tonozuka

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Glycoside Hydrolase Family GH63
Clan	GH-G
Mechanism	inverting
Active site residues	Inferred
CAZy DB link
https://www.cazy.org/GH63.html

Substrate specificities

Glycoside hydrolases of GH63 are exo-acting α-glucosidases. Eukaryotic members of this family are processing α-glucosidase I enzymes (mannosyl-oligosaccharide glucosidase, EC 3.2.1.106), which specifically hydrolyze the terminal α-1,2-glucosidic linkage in the N-linked oligosaccharide precursor, Glc₃Man₉GlcNAc₂, to produce β-glucose and Glc₂Man₉GlcNAc₂. Processing α-glucosidase I thus plays a critical role in the maturation of eukaryotic N-glycans. The enzymatic properties of Cwh41p, a processing α-glucosidase I from Saccharomyces cerevisiae, have been intensively studied [1] (also reviewed in [2]).

Genes encoding GH63 enzymes have also been found in archaea and bacteria, but their natural substrates are still unclear, as these organisms are not known to produce eukaryotic N-linked oligosacharides. A bacterial GH63 enzyme, Escherichia coli YgjK, demonstrated the highest activity toward the α-1,3-glucosidic linkage of nigerose (Glc-α-1,3-Glc) among the commercially available sugars tested, but the Km value for nigerose was substantially higher than that for other typical α-glucosidases [3]. In 2013, the substrates of GH63 enzymes from Thermus thermophilus HB27 and Rubrobacter radiotolerans RSPS-4 have been identified as α-D-mannopyranosyl-1,2-D-glycerate (mannosylglycerate) and α-D-glucopyranosyl-1,2-D-glycerate (glucosylglycerate) [4].

Kinetics and Mechanism

Family GH63 enzymes are inverting enzymes, as first shown by NMR on a processing α-glucosidase I from S. cerevisiae [5].

Catalytic Residues

The catalytic residues were inferred by comparing the catalytic (α/α)₆ barrel domain of the GH63 enzyme, E. coli YgjK, with those of GH15 and GH37 enzymes. In the case of GH37 and GH63, both of which belong to clan GH-G, the catalytic general acid is predicted as an Asp residue (Asp501 in E. coli YgjK), and the general base is considered to be a Glu residue (Glu727 in E. coli YgjK) [3]. Although both of the corresponding residues of GH15, which belongs to clan GH-L, are identified as Glu residues, the positions of the catalytic residues of GH15, GH37, and GH63 are highly conserved [3, 6].

Three-dimensional structures

The crystal structures of the bacterial GH63 proteins, E. coli YgjK [3] (multiple PDB entries) and Thermus thermophilus uncharacterised protein TTHA0978 (PDB 2z07), have been reported. The catalytic domain consists of an (α/α)₆ barrel fold. The main chain of the (α/α)₆ barrel domain shares high structural similarity with those of GH15, GH37, GH65, and GH94 [3, 6]. This similarity had been predicted on the basis of sequence comparison, before their crystal structures were available [7]. The first crystal structure of the eukaryotic processing α-glucosidase I (PDB 4j5t) has been reported in 2013 [8].

Family Firsts

First gene cloning

Human processing α-glucosidase I [9].

First stereochemistry determination

Processing α-glucosidase I from Saccharomyces cerevisiae (Cwh41p) [5].

First general acid residue identification

Inferred from structural comparison [3].

First general base residue identification

Inferred from structural comparison [3].

First 3-D structure

Escherichia coli YgjK, an enzyme showing the highest activity for the α-1,3-glucosidic linkage of nigerose [3].

First 3-D structure of a eukaryotic GH63 enzyme

A transmembrane-deleted form of processing α-glucosidase I from Saccharomyces cerevisiae [8].

References

1. Dhanawansa R, Faridmoayer A, van der Merwe G, Li YX, and Scaman CH. (2002). Overexpression, purification, and partial characterization of Saccharomyces cerevisiae processing alpha glucosidase I. Glycobiology. 2002;12(3):229-34. DOI:10.1093/glycob/12.3.229 |
2. Herscovics A (1999). Processing glycosidases of Saccharomyces cerevisiae. Biochim Biophys Acta. 1999;1426(2):275-85. DOI:10.1016/s0304-4165(98)00129-9 |
3. Kurakata Y, Uechi A, Yoshida H, Kamitori S, Sakano Y, Nishikawa A, and Tonozuka T. (2008). Structural insights into the substrate specificity and function of Escherichia coli K12 YgjK, a glucosidase belonging to the glycoside hydrolase family 63. J Mol Biol. 2008;381(1):116-28. DOI:10.1016/j.jmb.2008.05.061 |
4. Alarico S, Empadinhas N, and da Costa MS. (2013). A new bacterial hydrolase specific for the compatible solutes α-D-mannopyranosyl-(1→2)-D-glycerate and α-D-glucopyranosyl-(1→2)-D-glycerate. Enzyme Microb Technol. 2013;52(2):77-83. DOI:10.1016/j.enzmictec.2012.10.008 |
5. Palcic MM, Scaman CH, Otter A, Szpacenko A, Romaniouk A, Li YX, and Vijay IK. (1999). Processing alpha-glucosidase I is an inverting glycosidase. Glycoconj J. 1999;16(7):351-5. DOI:10.1023/a:1007096011392 |
6. Gibson RP, Gloster TM, Roberts S, Warren RA, Storch de Gracia I, García A, Chiara JL, and Davies GJ. (2007). Molecular basis for trehalase inhibition revealed by the structure of trehalase in complex with potent inhibitors. Angew Chem Int Ed Engl. 2007;46(22):4115-9. DOI:10.1002/anie.200604825 |
7. Stam MR, Blanc E, Coutinho PM, and Henrissat B. (2005). Evolutionary and mechanistic relationships between glycosidases acting on alpha- and beta-bonds. Carbohydr Res. 2005;340(18):2728-34. DOI:10.1016/j.carres.2005.09.018 |
8. Barker MK and Rose DR. (2013). Specificity of Processing α-glucosidase I is guided by the substrate conformation: crystallographic and in silico studies. J Biol Chem. 2013;288(19):13563-74. DOI:10.1074/jbc.M113.460436 |
9. Kalz-Füller B, Bieberich E, and Bause E. (1995). Cloning and expression of glucosidase I from human hippocampus. Eur J Biochem. 1995;231(2):344-51. DOI:10.1111/j.1432-1033.1995.tb20706.x |