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Glycoside Hydrolase Family 36

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Glycoside Hydrolase Family GH36
Clan GH-D
Mechanism retaining
Active site residues known
CAZy DB link

Substrate specificities

Glycoside hydrolases of family 36 exhibit α-galactosidase and α-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.

Kinetics and Mechanism

Family GH36 α-galactosidases are anomeric configuration-retaining enzymes, as first shown by NMR studies on the α-galactosidase GalA from Thermotoga maritima [1]. Correspondingly, GH36 enzymes use a classical Koshland double-displacement mechanism [2], like their GH27 relatives in Clan GH-D. This mechanism involves the formation of a covalent glycosyl-enzyme intermediate [3] that partitions predominantly to water in hydrolytic enzymes and to saccharide acceptor substrates in transglycosylating enzymes, such as stachyose and raffinose synthases.

Catalytic Residues

Detailed phylogenetic analysis of archaeal GH36 α-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 GH27 [4]. Mutagenesis of the corresponding residues in Sulfolobus solfataricus α-galactosidase GalS dramatically reduced enzyme activity: the D367G (catalytic nucleophile) and D425G (general acid/base) mutant had <1 x 10–3 and 5 x 10–3 lower activity than the wild type enzyme when assayed against p-nitrophenyl α-D-galactopyranoside [4]. Rescue of the catalytic function of both enzyme mutants was unsuccessful with both azide and formate anions [4].

The identities of the catalytic residues in GH36 were also confirmed in the Thermotoga maritima α-galactosidase GalA, guided by structural homology with GH27 enzymes [1]. 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 α-D-galactopyranoside and resulted in formation of β-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 α-D-galactopyranoside, while addition of azide resulted in formation of α-galactopyranosyl azide by nucleophilic attack on the β-linked glycosyl enzyme intermediate.

Three-dimensional structures

In June 2005, the first three-dimensional structural coordinates for a member of this family, α-galactosidase TmGalA from Thermotoga maritima, were deposited by the Joint Center For Structural Genomics (JCSG) (X-ray, 2.34 Å, PDB 1zy9) [5]. 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 [1, 6] and a number of conserved substrate-binding residues [1]. The active sites of both GH27 and GH36 enzymes are presented by (α/β)8 (TIM) barrel domains. Both GH36 and GH27 enzymes contain a C-terminal β-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 α-galactosidase, PDB 1uas). Notably, an extra N-terminal, primarily β-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 [1].

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" α-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 [4]. 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 [6].

Family Firsts

First sterochemistry determination
Thermotoga maritima α-galactosidase, by NMR [1].
First catalytic nucleophile identification
Sulfolobus solfataricus α-galactosidase GalS, by sequence homology with GH27 enzymes and mutagenesis [4]. Subsequently confirmed in Thermotoga maritima α-galactosidase by structural homology, mutagenesis, and azide rescue [1].
First general acid/base residue identification
Sulfolobus solfataricus α-galactosidase GalS, by sequence homology with GH27 enzymes and mutagenesis [4]. Subsequently confirmed in Thermotoga maritima α-galactosidase by structural homology, mutagenesis, and azide rescue [1].
First 3-D structure
Thermotoga maritima α-galactosidase by X-ray crystallography. Coordinates (PDB 1zy9) deposited in 2005 as part of a high-throughput functional genomics project [5], structural analyses published by other groups in 2006 and 2007 [1, 6].


  1. Comfort DA, Bobrov KS, Ivanen DR, Shabalin KA, Harris JM, Kulminskaya AA, Brumer H, and Kelly RM. Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases. Biochemistry. 2007 Mar 20;46(11):3319-30. DOI:10.1021/bi061521n | PubMed ID:17323919 | HubMed [Comfort2007]
  2. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006 [Sinnott1990]
  3. Vocadlo DJ, Davies GJ, Laine R, and Withers SG. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature. 2001 Aug 23;412(6849):835-8. DOI:10.1038/35090602 | PubMed ID:11518970 | HubMed [Vocadlo2001]
  4. Brouns SJ, Smits N, Wu H, Snijders AP, Wright PC, de Vos WM, and van der Oost J. Identification of a novel alpha-galactosidase from the hyperthermophilic archaeon Sulfolobus solfataricus. J Bacteriol. 2006 Apr;188(7):2392-9. DOI:10.1128/JB.188.7.2392-2399.2006 | PubMed ID:16547025 | HubMed [Brouns2006]
  5. Lesley SA, Kuhn P, Godzik A, Deacon AM, Mathews I, Kreusch A, Spraggon G, Klock HE, McMullan D, Shin T, Vincent J, Robb A, Brinen LS, Miller MD, McPhillips TM, Miller MA, Scheibe D, Canaves JM, Guda C, Jaroszewski L, Selby TL, Elsliger MA, Wooley J, Taylor SS, Hodgson KO, Wilson IA, Schultz PG, and Stevens RC. Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline. Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11664-9. DOI:10.1073/pnas.142413399 | PubMed ID:12193646 | HubMed [Lesley2002]
  6. Ernst HA, Lo Leggio L, Willemoës M, Leonard G, Blum P, and Larsen S. Structure of the Sulfolobus solfataricus alpha-glucosidase: implications for domain conservation and substrate recognition in GH31. J Mol Biol. 2006 May 12;358(4):1106-24. DOI:10.1016/j.jmb.2006.02.056 | PubMed ID:16580018 | HubMed [Ernst2006]
All Medline abstracts: PubMed | HubMed
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