Glycoside Hydrolase Family 27

• Author: ^^^Harry Brumer^^^
• Responsible Curator: ^^^Harry Brumer^^^

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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

Glycoside hydrolases of family 27 possess a range of activities. Alpha-galactosidase activity has been observed in both bacterial and eukaryotic members of GH27, whereas 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 Fabry and Schindler 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 general 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 known point mutations in human alpha-galactosidase A that 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].

Three-dimensional structures

Published in 2002, the 3-D structure of the chicken (Gallus gallus) N-acetylgalactosaminidase solved by Garman et al. using X-ray crystallography (1.9 Å resolution) represented the first structure of an enzyme from GH27, and indeed Clan GH-D [9]. Futhermore, the simultaneous solution of an enzyme-product complex (2.4 Å), was instrumental in defining the catalytic acid/base residue in this GH family and clan [9], as described above. Soon thereafter, structures of the rice (Oryza sativa) alpha-galactosidase (2003) [10], human (Homo sapiens) alpha-galactosidase A (2004) [11], and Hypocrea jecorina (née Trichoderma reesei) alpha-galactosidase (2004) [12] were presented in both free and product-complexed forms. All of these structures indicated that GH27 enzymes are comprised of an N-terminal (alpha/beta)8 (TIM) barrel domain and a C-terminal anti-parallel beta-jellyroll domain, the former of which contains the enzyme catalytic center composed by loop residues at the ends of beta strands 1-7.

The known overall structures of GH27 enzymes are all highly conserved and the N-terminal domains are all closely superimposable, with minor exceptions including the H. jecorina (T. reesei) alpha-galactosidase [12], which contains a 40 amino acid insertion in loop beta4-alpha4, and the animal enzymes [9, 11], which contain a short 10 residue insertion in the alpha1-beta1 loop [1]. The C-terminal domains, although similar, are less well conserved, both at the primary and tertiary structural levels [1]. In keeping with the exo mode of action of these enzymes, which cleave alpha-Gal from the non-reducing terminii of their substrates, the active sites are pocket-shaped [9, 10, 11, 12]. Specificity for the 2-hydroxyl substituent, in the case of alpha-galactosidases in the family, and the 2-deoxy-2-N-acetyl substituent, in the case of the alpha-N-acetylgalactosaminidases, is dictated by the presence of correspondingly large or small active-site binding residues, respectively [11] (reviewed in [1]). Based on these observations, phylogenetic analysis has been presented which may have some power to predict specificity within GH27 [11].

As predicted by their common membership in Clan GH-D, GH36 enzymes likewise present active sites on (alpha/beta)8 barrel domains [6]. GH36 enzymes also contain a related C-terminal beta-sheet domain, in addition to a large beta-supersandwich N-terminal domain not found in GH27 enzymes [6]. Structural analysis of a GH31 enzyme has led to the addition of this family to Clan GH-D [13].

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 enzymic 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 |
13. Ernst HA, Lo Leggio L, Willemoës M, Leonard G, Blum P, and Larsen S. (2006). Structure of the Sulfolobus solfataricus alpha-glucosidase: implications for domain conservation and substrate recognition in GH31. J Mol Biol. 2006;358(4):1106-24. DOI:10.1016/j.jmb.2006.02.056 |