Crystal Structure of α-Galactosidase from Trichoderma reesei and Its Complex with Galactose: Implications for Catalytic Mechanism

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Abstract

The crystal structures of α-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, β-d-galactose, have been determined at 1.54 Å and 2.0 Å resolution, respectively. The α-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the α-galactosidase consists of two domains, an N-terminal catalytic domain of the (β/α)8 barrel topology and a C-terminal domain which is formed by an antiparallel β-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the α-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the α-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the α-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.

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)

  • J.S. Kim et al.

    Crystal structure of a maltogenic amylase provides insights into a catalytic versatility

    J. Biol. Chem.

    (1999)
  • S. Elgavish et al.

    Lectin-carbohydrate interactions: different folds, common recognition principles

    Trends Biochem. Sci.

    (1997)
  • A.R. Kolatkar et al.

    Mechanism of N-acetylgalactosamine binding to a C-type animal lectin carbohydrate-recognition domain

    J. Biol. Chem.

    (1998)
  • S. Elgavish et al.

    Structures of the Erythrina corallodendron lectin and of its complexes with mono- and disaccharides

    J. Mol. Biol.

    (1998)
  • Z. Otwinowski et al.

    Processing of X-ray diffraction data collected in oscillation mode

    Methods Enzymol.

    (1997)
  • E. de La Fortelle et al.

    Maximum-likelihood heavy-atom parameter refinement for the multiple isomorphous replacement and multiwavelength anomalous diffraction methods

    Methods Enzymol.

    (1997)
  • D.E. McRee

    XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density

    J. Struct. Biol.

    (1999)
  • E.A. Merritt et al.

    Raster3D photorealistic molecular graphics

    Methods Enzymol.

    (1997)
  • D.P. Germain et al.

    Fabry disease: twenty novel α-galactosidase A mutations and genotype-phenotype correlations in classical and variant phenotypes

    Mol. Med.

    (2002)
  • P.M. Coutinho et al.

    Carbohydrate-active enzymes: an integrated database approach

  • A.M. Kachurin et al.

    Role of methionine in the active site of α-galactosidase from Trichoderma reesei

    Biochem. J.

    (1995)
  • Z. Dauter et al.

    Entering a new phase: using solvent halide ions in protein structure determination

    Structure

    (2001)
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