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Glycoside Hydrolase Family 27

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Glycoside Hydrolase Family GH27
Clan GH-D
Mechanism retaining
Active site residues known
CAZy DB link

Substrate specificities

Glycoside hydrolases of family 27 possess a range of activities. α-Galactosidase activity has been observed in both bacterial and eukaryotic members of GH27, whereas α-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 α-galactosidase A and human α-N-acetylgalactosaminidase (also known as α-galactosidase B), defects in which produce the phenotypes associated with Fabry and Schindler lysosomal storage disorders, respectively [1, 2]. Guided by protein tertiary structural analysis (vide infra), the substrate specificities of the human α-galactosidase A and human α-N-acetylgalactosaminidase have been interconverted by protein engineering [3].

Kinetics and Mechanism

Family GH27 α-galactosidases are anomeric configuration-retaining enzymes, as first demonstrated by proton NMR studies of the hydrolysis of p-nitrophenyl α-galactopyranoside by an α-galactosidase isolated from the white-rot fungus Phanerochaete chrysosporium [4]. GH27 enzymes are thus expected to use a classical Koshland double-displacement mechanism [5], which involves the formation of a covalent glycosyl-enzyme intermediate [6]. As predicted based on their common clanship in Clan GH-D, Glycoside Hydrolase Family 36 (GH36) enzymes also operate through the same "retaining" mechanism [7].

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 α-galactosidase by labelling with 2',4',6'-trinitrophenyl 2-deoxy-2,2-difluoro-α-D-lyxo-hexopyranoside ("2,2-difluoro-α-galactosyl picrate") [8] only slightly predated the identification of the same conserved aspartate in the green coffee bean α-galactosidase (Asp-145 in the sequence LKYDNCNNN) using 5-fluoro-α-D-galactopyranosyl fluoride as a labelling agent [9]. The subsequent observation of a trapped covalent 2-deoxy-2,2-difluorogalactosyl-enzyme intermediate human (Homo sapiens) α-galactosidase A on Asp170 of that enzyme provided further "visual" confirmation of the identity of the catalytic nucleophile in this family [10].

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 [11]. 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 α-galactosidase [12], human α-galactosidase A [13], and the Hypocrea jecorina (née Trichoderma reesei) α-galactosidase [14] have similarly implicated the homologous residue in these enzymes in catalysis.

Interestingly, of the over 200 known point mutations in human α-galactosidase A that lead to Fabry disease, very few involve the catalytic residues [1, 11, 13]. 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, 13]. Several other mutations are known to affect key active site structural or substrate-binding residues in human α-galactosidase A [13]. 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 α-galactosidase A [13] and chicken N-acetylgalactosaminidase [11] 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 [11]. 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 [11], as described above. Soon thereafter, structures of the rice (Oryza sativa) α-galactosidase (2003) [12], human (Homo sapiens) α-galactosidase A (2004) [13], and Hypocrea jecorina (née Trichoderma reesei) α-galactosidase (2004) [14] were presented in both free and product-complexed forms. All of these structures indicated that GH27 enzymes are comprised of an N-terminal (α/β)8 (TIM) barrel domain and a C-terminal anti-parallel β-jellyroll domain, the former of which contains the enzyme catalytic center composed by loop residues at the ends of β strands 1-7.

In 2010, Garman and co-workers published an ensemble of tertiary structures of the human α-galactosidase A, which included a trapped covalent 2-deoxy-2,2-difluorogalactosyl-enzyme intermediate in an 1S3 skew boat conformation [10]. Together with native, substrate-bound (E•S, Michaelis complex), and product-bound (E•P) structures, this work provided the first complete analysis of substrate distortion along the enzyme reaction coordinate in GH27. Thus, GH27 enzymes use a 4C1 ↔ (4H3)1S3 ↔ (4H3)4C1 conformational itinerary. This is the same itinerary employed by GH31 enzymes (also of Clan GH-D, vide infra), while the α-l-fucosidases of GH29 use the mirror-image itinerary: 1C4 ↔ (3H4)3S1 ↔ (3H4)1C4 (see [15] for a full discussion).

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) α-galactosidase [14], which contains a 40 amino acid insertion in loop β4-α4, and the animal enzymes [11, 13], which contain a short 10 residue insertion in the α1-β1 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 α-Gal from the non-reducing termini of their substrates, the active sites are pocket-shaped [11, 12, 13, 14]. Specificity for the 2-hydroxyl substituent, in the case of α-galactosidases in the family, and the 2-deoxy-2-N-acetyl substituent, in the case of the α-N-acetylgalactosaminidases, is dictated by the presence of correspondingly large or small active-site binding residues, respectively [13] (reviewed in [1]). Based on these observations, phylogenetic analysis has been presented which may have some power to predict specificity within GH27 [13].

As predicted by their common membership in Clan GH-D, GH36 enzymes likewise present active sites on (α/β)8 barrel domains [7]. GH36 enzymes also contain a related C-terminal β-sheet domain, in addition to a large β-supersandwich N-terminal domain not found in GH27 enzymes [7]. Structural analysis of a GH31 enzyme has led to the addition of this family to Clan GH-D [16].

Family Firsts

First sterochemistry determination
Retention of anomeric stereochemistry demonstrated by H-1 NMR for the main α-galactosidase from the white-rot fungus Phanerochaete chrysosporium [4].
First catalytic nucleophile identification
Phanerochaete chrysosporium α-galactosidase by mechanism-based labelling with 2',4',6'-trinitrophenyl 2-deoxy-2,2-difluoro-α-D-lyxo-hexopyranoside ("2,2-difluoro-α-galactosyl picrate"), pepsin digestion, and mass spectrometry [8].
First general acid/base residue identification
Chicken (Gallus gallus) N-acetylgalactosaminidase by X-ray structural analysis of an enzyme-N-acetylgalactosamine complex [11].
First 3-D structure
Chicken N-acetylgalactosaminidase, both free enzyme and in complex with N-acetylgalactosamine [11].


  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 | PubMed ID:17391432 [5]
  3. Tomasic IB, Metcalf MC, Guce AI, Clark NE, and Garman SC. (2010). Interconversion of the specificities of human lysosomal enzymes associated with Fabry and Schindler diseases. J Biol Chem. 2010;285(28):21560-6. DOI:10.1074/jbc.M110.118588 | PubMed ID:20444686 [Tomasic2010]
  4. 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 PubMed ID:10085226 [1]
  5. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006

  6. 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 | PubMed ID:11518970 [7]
  7. 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 | PubMed ID:17323919 [8]
  8. 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 | PubMed ID:10933800 [2]
  9. 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 | PubMed ID:11128583 [9]
  10. Guce AI, Clark NE, Salgado EN, Ivanen DR, Kulminskaya AA, Brumer H 3rd, and Garman SC. (2010). Catalytic mechanism of human alpha-galactosidase. J Biol Chem. 2010;285(6):3625-3632. DOI:10.1074/jbc.M109.060145 | PubMed ID:19940122 [GuceJBC2010]
  11. 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 | PubMed ID:12005440 [3]
  12. 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 | PubMed ID:12657636 [11]
  13. 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 | PubMed ID:15003450 [10]
  14. 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 | PubMed ID:15136043 [12]
  15. Lammerts van Bueren A, Ardèvol A, Fayers-Kerr J, Luo B, Zhang Y, Sollogoub M, Blériot Y, Rovira C, and Davies GJ. (2010). Analysis of the reaction coordinate of alpha-L-fucosidases: a combined structural and quantum mechanical approach. J Am Chem Soc. 2010;132(6):1804-6. DOI:10.1021/ja908908q | PubMed ID:20092273 [LammertsJACS2010]
  16. 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 | PubMed ID:16580018 [13]

All Medline abstracts: PubMed