Structure analysis of a glycosides hydrolase family 42 cold-adapted beta-galactosidase from Rahnella sp R3

摘要

The beta-galactosidase isolated from a psychrotrophic bacterium, Rahnella sp. R3 (R-beta-Gal), exhibits high activity at low temperature and has potential in the dairy industry. R-beta-Gal is a member of the glycoside hydrolases family 42 (GH42), and it forms a 225 kDa trimeric structure in solution. The crystals of R-beta-Gal were acquired via hanging-drop vapor-diffusion method, and the X-ray crystal structure of the native R-beta-Gal was determined at a 2.5 angstrom resolution. It is the first structure of a cold-adapted GH42 enzyme. In the crystallographic asymmetric unit, there were two homotrimers of the enzyme. Each monomer consists of three domains, an N-terminal catalytic domain which is a (beta/alpha) 8 barrel, a mixed beta-sheet and a-helices domain, and a C-terminal beta-sandwich domain. Two putative residues might be involved in catalysis, a proton donor E157 and a nucleophile E314, were superimposed well with the catalytic residues of other beta-galactosidases. Site-directed mutagenesis targeting these residues abolished the activity of the enzyme. Structure and sequence comparison of R-beta-Gal with two mesophilic beta-gals and a thermophilic beta-gal indicated that intramolecular force and higher structural flexibility might result in the cold-adaptation of R-beta-Gal.