Glycoside Hydrolase Family 27

Glycoside Hydrolase Family GH27
Clan	GH-D
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH27.html

Substrate specificities

Alpha-galactosidase activity has been observed in both bacterial and eukaryotic members of GH27, while alpha-N-acetylgalactosaminidase activity has been observed in certain eukaryotic enzymes, including human, mouse, and chicken. Bacterial GH27 isomaltodextranases have also been identified. Notably, this family contains both human alpha-galactosidase A and human alpha-N-acetylgalactosaminidase (also known as alpha-galactosidase B), defects in which produce the phenotypes associated with Schindler and Fabry lysosomal storage disorders, respectively [1, 2].

Kinetics and Mechanism

Family GH27 alpha-galactosidases are anomeric configuration-retaining enzymes, as first demonstrated by proton NMR studies of the hydrolysis of p-nitrophenyl alpha-galactopyranoside by an alpha-galactosidase isolated from the white-rot fungus Phanerochaete chrysosporium [3]. GH27 enzymes are thus expected to use a classical Koshland double-displacement mechanism [4], which involves the formation of a covalent glycosyl-enzyme intermediate [5]. As predicted based on their common clanship in Clan GH-D, Glycoside Hydrolase Family 36 (GH36) enzymes also operate through the same "retaining" mechanism [6].

Catalytic Residues

The conserved amino acid sidechain that functions as the catalytic nucleophile in GH27 has been identified in two different eukaryotic family members by mechanism-based labelling, proteolytic digestion, and mass spectrometric analysis. Identification of Asp-130 in the YLKYDNC sequence fragment of the Phanerochaete chrysosporium alpha-galactosidase by labelling with 2',4',6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D-lyxo-hexopyranoside ("2,2-difluoro-alpha-galactosyl picrate") [7] only slightly predated the identification of the same conserved aspartate in the green coffee bean alpha-galactosidase (Asp-145 in the sequence LKYDNCNNN) using 5-fluoro-alpha-D-galactopyranosyl fluoride as a labelling agent [8].

The catalytic acid/base residue in this family was first identified by X-ray structural analysis of the chicken (Gallus gallus) N-acetylgalactosaminidase in complex with N-acetylgalactosamine [9]. The position of the product within the enzyme active site indicated that Asp-201 in the sequence CNLWRNYDDIQDSW was the obvious candidate to fulfill this role. Subsequent product complexes of the rice alpha-galactosidase [10], human alpha-galactosidase A [11], and the Hypocrea jecorina (née Trichoderma reesei) alpha-galactosidase [12] have similarly implicated the homologous residue in these enzymes in catalysis.

Interestingly, of the over 200 point mutations in human alpha-galactosidase A which lead to Fabry disease, very few involve the catalytic residues [1, 9, 11]. While many mutations are thought to disrupt the hydrophobic core of the enzyme or otherwise disrupt protein folding, only the D170V, D170H, and D231N genotypic variants are known, with obvious catalytic implications [1, 11]. Several other mutations are known to affect key active site structural or substrate-binding residues in human alpha-galactosidase A [11]. Whereas Fabry disease is X-linked and therefore comparatively more common, the autosomal recessive Schindler disease is rare [1]. Comparative analysis using the structurally similar human alpha-galactosidase A [11] and chicken N-acetylgalactosaminidase [9] enzymes has indicated that none of the few known mutations in the human GH27 N-acetylgalactosaminidase occur in the catalytic nor active site residues [1].

Family Firsts

First sterochemistry determination

Retention of anomeric stereochemistry demonstrated by H-1 NMR for the main alpha-galactosidase from the white-rot fungus Phanerochaete chrysosporium [3].

First catalytic nucleophile identification

Phanerochaete chrysosporium alpha-galactosidase by mechanism-based labelling with 2',4',6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D-lyxo-hexopyranoside ("2,2-difluoro-alpha-galactosyl picrate"), pepsin digestion, and mass spectrometry [7].

First general acid/base residue identification

Chicken (Gallus gallus) N-acetylgalactosaminidase by X-ray structural analysis of an enzyme-N-acetylgalactosamine complex [9].

First 3-D structure

Chicken N-acetylgalactosaminidase, both free enzyme and in complex with N-acetylgalactosamine [9].

References

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1. Garman, S.C. (2006) Structural studies on alpha-GAL and alpha-NAGAL: The atomic basis of Fabry and Schindler diseases. Biocatalysis and Biotransformation 24, 129-136. DOI: 10.1080/10242420600598194

4. Sinnott, M.L. (1990) Catalytic mechanisms of enzymatic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006

5. Vocadlo DJ, Davies GJ, Laine R, and Withers SG. (2001). Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature. 2001;412(6849):835-8. DOI:10.1038/35090602 |
6. Comfort DA, Bobrov KS, Ivanen DR, Shabalin KA, Harris JM, Kulminskaya AA, Brumer H, and Kelly RM. (2007). Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases. Biochemistry. 2007;46(11):3319-30. DOI:10.1021/bi061521n |