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

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.