Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism.
The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-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 alpha-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 alpha-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 alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.
Golubev
A M
AM
Petersburg Nuclear Physics Institute, Gatchina, St Petersburg, 188300, Russia.
Nagem
R A P
RA
Brandão Neto
J R
JR
Neustroev
K N
KN
Eneyskaya
E V
EV
Kulminskaya
A A
AA
Shabalin
K A
KA
Savel'ev
A N
AN
Polikarpov
I
I
D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D005690:Galactose / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D014242:Trichoderma / Q000201:enzymology*; D000519:alpha-Galactosidase / Q000737:chemistry / Q000378:metabolism*
D000519:alpha-Galactosidase; D005690:Galactose