Journal of Molecular Biology
Crystal Structure of α-Galactosidase from Trichoderma reesei and Its Complex with Galactose: Implications for Catalytic Mechanism
Introduction
α-Galactosidase (α-d-galactoside galactohydrolase; melibiase; EC 3.2.1.22) is an enzyme that catalyzes the hydrolytic cleavage of terminal α-d-galactopyranosyl residues from galactomannanes and oligosaccharides such as melibiose, raffinose and stachyose. α-Galactosidases have been isolated from various sources, including bacteria, fungi, plants and animals. In humans, mutations in the α-GAL A gene located at the Xq22 chromosome lead to Fabry disease, a lysosomal storage disorder, caused by defects in oligosaccharide catabolism. Deposits of glycosphingolipids with terminal α-galactose in vessel walls and various organs correlate with the pathology.1
All known glycoside hydrolases are divided into 87 families according to the similarities of their amino acid sequences.2 Within this classification, all such enzymes can be separated into two groups on the basis of the possible catalytic mechanisms for glycoside hydrolase action: (i) those that act with retention of the stereochemistry at the substrate anomeric center; and (ii) those that induce its inversion.3 Both mechanisms employ two side-chain carboxylate groups of Asp or Glu residues in the active site to mediate catalysis. The inverting glycosyl hydrolases catalyze glycosidic bond cleavage by a one-step mechanism, where one of the active-site carboxylate groups acts as a general base catalyst to activate a nucleophilic water molecule, whereas the second plays the role of a general acid catalyst to protonate the leaving group on departure. The retaining enzymes act via a double-displacement mechanism, wherein one of the catalytic carboxylate groups operates as a nucleophile to generate a glycosyl–enzyme intermediate. The second carboxylate group acts, in turn, as a general acid and general base catalyst to promote the formation and breakdown, respectively, of the intermediate. On the basis of amino acid sequence similarities, all known α-galactosidases have been classified into glycoside hydrolase families 4, 27, 36 and 57.2
The Trichoderma reesei α-GAL shows strong similarities in its sequence and enzymatic properties to the GHF 27 carbohydrases.4., 5. This family includes enzymes of three types: α-galactosidases, α-N-acetylgalactosaminidases and isomalto-dextranases. The catalytic activity of the enzymes from GHF 27 is based on a similar double-displacement mechanism. Nothing or little is known about the catalytic mechanism of α-galactosidases from families 4, 36 and 57. Recently, crystallographic structures of two carbohydrases from GHF 27, chicken α-N-acetylgalactosaminidase and rice α-galactosidase were reported.6., 7. Both of them are consistent with a double-displacement mechanism of reaction.
The α-galactosidase isolated from the mesophilic fungus T. reesei is a glycoprotein with average molecular mass of 54 kDa. Purification, enzymatic properties and crystallization of the protein have been described.5., 8. Oxidative activation was reported for T. reesei α-GAL, when its enzymatic activity toward PNPG increases 12 times after treatment of the enzyme with non-specific oxidants. It was shown that specific oxidation of a single methionine residue, out of five available in the protein, to methionine sulphoxide caused the activation effect. Galactose prevents the oxidative activation, which suggest that the modified methionine residue may lie near the active site.9
Here, we report the structures of the T. reesei α-galactosidase determined at 1.54 Å resolution and its complex with β-d-galactose refined at 2.0 Å resolution. The crystallographic structures reveal the mode of action of the enzyme. Furthermore, they reveal the glycosylation of the enzyme and offer a plausible explanation for the mechanism of oxidative activation as well as the kinetic peculiarities of the enzyme.
Section snippets
Quality of the model and three-dimensional structure of the α-galactosidase
The structure of α-galactosidase was determined using isomorphous and anomalous differences provided by a single cesium derivative (SIRAS method) obtained by the quick cryo-soaking technique.10., 11., 12. The statistics for data collection of the native α-Gal, Cs derivative and enzyme–galactose complex are shown in Table 1. The final model for the uncomplexed structure has an R-factor of 0.152 (Rfree 0.185) to 1.54 Å resolution. The rms deviations from ideal values are 0.010 Å for bond lengths
Crystallization, derivative preparation and data collection
The crystals of α-galactosidase were grown by the hanging-drop method as described.7 The crystals belong to the orthorhombic space group P212121 (Table 1). The crystal complex of α-galactosidase-inhibitor was obtained by crystallization under the same conditions in the presence of 10 mM d-galactose. The crystals of the complex belong to the same space group as the native α-galactosidase crystals but have different unit cell dimensions (Table 1).
A large number of heavy-atom salts were tested for
Acknowledgements
This work was supported by grant 302125/02-7 from CNPq (Conselho Nacional de Desenvolmento Cientifico e Tecnologico), grants 99/03387-4 and 02/14208-8 from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), grant 03-04-48756 from the Russian Foundation for Basic Research, and a grant from the Program for Basic Research “Physical-Chemical Biology” from the Presidium of Russian Academy of Sciences.
References (45)
- et al.
Structures and mechanisms of glycosyl hydrolases
Structure
(1995) - et al.
Transglycosylation activity of α-d-galactosidase from Trichoderma reesei. An investigation of the active site
Carbohydr. Res.
(1997) - et al.
Enzymatic properties of α-galactosidase from Trichoderma reesei in the hydrolysis of galactooligosaccharides
Enzyme Microb. Technol.
(2002) - et al.
The 1.9 Å structure of α-N-acetylgalactosaminidase: molecular basis of glycosidase deficiency diseases
Structure
(2002) - et al.
Crystal structure of rice α-galactosidase complexed with d-galactose
J. Biol. Chem.
(2003) - et al.
Crystallization of alpha-galactosidase from Trichoderma reesei
J. Mol. Biol.
(1993) - et al.
Phasing on rapidly soaked ions
Methods Enzymol.
(2003) - et al.
Crystal structure of Thermoactinomyces vulgaris R-47 α-amylase II (TVAII) hydrolyzing cyclodextrins and pullulan at 2.6 Å resolution
J. Mol. Biol.
(1999) - et al.
Glycoside hydrolases and glycosyltransferases: families and functional modules
Curr. Opin. Struct. Biol.
(2001) - et al.
Crystal structures of a mutant maltotetraose-forming exo-amylase cocrystallized with maltopentaose
J. Mol. Biol.
(1997)
Crystal structure of a maltogenic amylase provides insights into a catalytic versatility
J. Biol. Chem.
Lectin-carbohydrate interactions: different folds, common recognition principles
Trends Biochem. Sci.
Mechanism of N-acetylgalactosamine binding to a C-type animal lectin carbohydrate-recognition domain
J. Biol. Chem.
Structures of the Erythrina corallodendron lectin and of its complexes with mono- and disaccharides
J. Mol. Biol.
Processing of X-ray diffraction data collected in oscillation mode
Methods Enzymol.
Maximum-likelihood heavy-atom parameter refinement for the multiple isomorphous replacement and multiwavelength anomalous diffraction methods
Methods Enzymol.
XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density
J. Struct. Biol.
Raster3D photorealistic molecular graphics
Methods Enzymol.
Fabry disease: twenty novel α-galactosidase A mutations and genotype-phenotype correlations in classical and variant phenotypes
Mol. Med.
Carbohydrate-active enzymes: an integrated database approach
Role of methionine in the active site of α-galactosidase from Trichoderma reesei
Biochem. J.
Entering a new phase: using solvent halide ions in protein structure determination
Structure
Cited by (72)
Characterization of a high performance α-galactosidase from Irpex lacteus and its usage in removal of raffinose family oligosaccharides from soymilk
2019, International Journal of Biological MacromoleculesLow molecular weight α-galactosidase from black gram (Vigna mungo): Purification and insights towards biochemical and biophysical properties
2018, International Journal of Biological MacromoleculesBiochemical and structural characterization of Penicillium purpurogenum α-D galactosidase: Binding of galactose to an alternative pocket may explain enzyme inhibition
2017, Carbohydrate ResearchCitation Excerpt :This feature is common for almost all GH27 galactosidases [49], as this Trp is conserved. In GALP1, Trp56 is close to the galactose unit (2.9 Å) as it is in other enzymes with known structure; nevertheless, in the enzyme used as template the equivalent tryptophan (Trp37) is far from the galactose (>5.0 Å) and hydrophobic interaction with galactose is not possible [25]. In the case of GalαpNP as ligand, a short distance (2.8 Å) between the carboxylic group of Asp225 and the hydroxyl group of galactose-O1 was observed (Fig. 5B), similar to the case of melibiose.
Biochemical characterization of a novel thermophilic α-galactosidase from Talaromyces leycettanus JCM12802 with significant transglycosylation activity
2016, Journal of Bioscience and Bioengineering