Identification and molecular characterization of a novel type of alpha-galactosidase from Pyrococcus furiosus

An -galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first -galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other -galactosidases. Highest homology was found with -amylases classified in family 57 of glycoside hydrolases. The 364 amino acid protein had a calculated mass of 41.6 kDa. The recombinant -galactosidase specifically catalyzed the hydrolysis of para-nitrophenyl--galactopyranoside, and to some extent that of melibiose and raffinose. The enzyme proved to be an extremely thermo-active and thermostable -galactosidase with a temperature optimum of 115°C and a half-life time of 15 hours at 100°C. The pH optimum is between 5.0 and 5.5. Sequence analysis showed four conserved carboxylic residues. Site-directed mutagenesis was applied to identify the potential catalytic residues. Glu117Ala showed decreased enzyme activity, which could be rescued by the addition of azide or formate. It is concluded that glutamate 117 is the catalytic nucleophile, whereas the acid/base catalyst remains to be identified