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 diseases, 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.

-- COMMENT on mutations in these residues in diseases.

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

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

2. Garman SC (2007). Structure-function relationships in alpha-galactosidase A. Acta Paediatr. 2007;96(455):6-16. DOI:10.1111/j.1651-2227.2007.00198.x |
3. Brumer H 3rd, Sims PF, and Sinnott ML. (1999). Lignocellulose degradation by Phanerochaete chrysosporium: purification and characterization of the main alpha-galactosidase. Biochem J. 1999;339 ( Pt 1)(Pt 1):43-53. | Google Books | Open Library

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 |
7. Hart DO, He S, Chany CJ 2nd, Withers SG, Sims PF, Sinnott ML, and Brumer H 3rd. (2000). Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase. Biochemistry. 2000;39(32):9826-36. DOI:10.1021/bi0008074 |
8. Ly HD, Howard S, Shum K, He S, Zhu A, and Withers SG. (2000). The synthesis, testing and use of 5-fluoro-alpha-D-galactosyl fluoride to trap an intermediate on green coffee bean alpha-galactosidase and identify the catalytic nucleophile. Carbohydr Res. 2000;329(3):539-47. DOI:10.1016/s0008-6215(00)00214-7 |
9. Garman SC, Hannick L, Zhu A, and Garboczi DN. (2002). The 1.9 A structure of alpha-N-acetylgalactosaminidase: molecular basis of glycosidase deficiency diseases. Structure. 2002;10(3):425-34. DOI:10.1016/s0969-2126(02)00726-8 |
10. Fujimoto Z, Kaneko S, Momma M, Kobayashi H, and Mizuno H. (2003). Crystal structure of rice alpha-galactosidase complexed with D-galactose. J Biol Chem. 2003;278(22):20313-8. DOI:10.1074/jbc.M302292200 |
11. Garman SC and Garboczi DN. (2004). The molecular defect leading to Fabry disease: structure of human alpha-galactosidase. J Mol Biol. 2004;337(2):319-35. DOI:10.1016/j.jmb.2004.01.035 |
12. Golubev AM, Nagem RA, Brandão Neto JR, Neustroev KN, Eneyskaya EV, Kulminskaya AA, Shabalin KA, Savel'ev AN, and Polikarpov I. (2004). Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism. J Mol Biol. 2004;339(2):413-22. DOI:10.1016/j.jmb.2004.03.062 |