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Glycoside Hydrolase Family 63

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Glycoside Hydrolase Family GH63
Clan GH-G
Mechanism inverting
Active site residues Inferred
CAZy DB link

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, 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]. The aglycon specificity of YgjK was screened using its glycosynthase mutants (D324N and E727A), which synthesized 2-O-α-glucopyranosylgalactose from β-glucopyranosyl fluoride donor and galactose acceptor [4].

In 2013, the substrates of GH63 enzymes from Thermus thermophilus HB27 and Rubrobacter radiotolerans RSPS-4 were identified as compatible solutes, α-D-mannopyranosyl-1,2-D-glycerate (mannosylglycerate) and α-D-glucopyranosyl-1,2-D-glycerate (glucosylglycerate) [5]. Subsequently, glucosylglycerate hydrolase was identified in Mycobacterium hassiacum and was found to be involved in the recovery process from nitrogen starvation by hydrolyzing glucosylglycerate [6].

An orthologous gene for mannosyl/glucosylglycerate hydrolase was also found in the genome of plant Selaginella moellendorffii, and the gene product hydrolyzed these compatible solutes [7].

Kinetics and Mechanism

Family GH63 enzymes are inverting enzymes, as first shown by NMR on a processing α-glucosidase I from S. cerevisiae [8].

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, 9].

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, 9]. This similarity had been predicted on the basis of sequence comparison, before their crystal structures were available [10]. The first crystal structure of the eukaryotic processing α-glucosidase I (PDB 4j5t) has been reported in 2013 [11].

Family Firsts

First gene cloning
Human processing α-glucosidase I [12].
First stereochemistry determination
Processing α-glucosidase I from Saccharomyces cerevisiae (Cwh41p) [8].
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 [11].


  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 | PubMed ID:11971867 [Dhanawansa2002]
  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 | PubMed ID:9878780 [Herscovics1999]
  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 | PubMed ID:18586271 [Kurakata2008]
  4. Miyazaki T, Ichikawa M, Yokoi G, Kitaoka M, Mori H, Kitano Y, Nishikawa A, and Tonozuka T. (2013). Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity. FEBS J. 2013;280(18):4560-71. DOI:10.1111/febs.12424 | PubMed ID:23826932 [Miyazaki2013]
  5. 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 | PubMed ID:23273275 [Alarico2013]
  6. Alarico S, Costa M, Sousa MS, Maranha A, Lourenço EC, Faria TQ, Ventura MR, and Empadinhas N. (2014). Mycobacterium hassiacum recovers from nitrogen starvation with up-regulation of a novel glucosylglycerate hydrolase and depletion of the accumulated glucosylglycerate. Sci Rep. 2014;4:6766. DOI:10.1038/srep06766 | PubMed ID:25341489 [Alarico2014]
  7. Nobre A, Empadinhas N, Nobre MF, Lourenço EC, Maycock C, Ventura MR, Mingote A, and da Costa MS. (2013). The plant Selaginella moellendorffii possesses enzymes for synthesis and hydrolysis of the compatible solutes mannosylglycerate and glucosylglycerate. Planta. 2013;237(3):891-901. DOI:10.1007/s00425-012-1808-6 | PubMed ID:23179444 [Nobre2013]
  8. 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 | PubMed ID:10619707 [Palcic1999]
  9. 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 | PubMed ID:17455176 [Gibson2007]
  10. 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 | PubMed ID:16226731 [Stam2005]
  11. 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 | PubMed ID:23536181 [Barker2013]
  12. 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 | PubMed ID:7635146 [Kalz-Fuller1995]

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