CRYSTAL-STRUCTURE OF BETA-GLUCOSIDASE-A FROM BACILLUS-POLYMYXA - INSIGHTS INTO THE CATALYTIC ACTIVITY IN FAMILY-1 GLYCOSYL HYDROLASES

Family 1 glycosyl hydrolases are a very relevant group of enzymes beca use of the diversity of biological roles in which they are involved, a nd their generalized occurrence in all sorts of Living organisms. The biological plasticity of these enzymes is a consequence of the variety of beta-glycosidic substrates that they can hydrolyze: disaccharides such as cellobiose and lactose, phosphorylated disaccharides, cyanogen ic glycosides, etc. The crystal structure of BglA, a member of the fam ily, has been determined in the native state and complexed with glucon ate ligand, at 2.4 Angstrom and 2.3 Angstrom resolution, respectively. The subunits of the octameric enzyme display the (alpha/beta)(8) barr el structural fold previously reported for other family 1 enzymes. How ever, significant structural differences have been encountered in the loops surrounding the active-center cavity. These differences make a w ide and extended cavity in BglA, which seems to be able to accommodate substrates longer than cellobiose, its natural substrate. Furthermore , a third sub-site is encountered, which might have some connection wi th the transglycosylating activity associated to this enzyme and its c ertain activity against beta-1,4 oligosaccharides composed of more tha n two units of glucose. The particular geometry of the cavity which co ntains the active center of BglA must therefore account for both, hydr olytic and transglycosylating activities. A potent and well known inhi bitor of different glycosidases, D-glucono-1,5-lactone, was used in an attempt to define interactions of the substrate with specific protein residues. Although the lactone has transformed into gluconate under c rystallizing conditions, the open species still binds the enzyme, the conformation of its chain mimicking the true inhibitor. From the analy sis of the enzyme-ligand hydrogen bonding interactions, a detailed pic ture of the active center can be drawn, for a family 1 enzyme. Ln this way, GLn20, His121, Tyr296, Glu405 and Trp406 are identified as deter minant residues in the recognition of the substrate. In particular, tw o bidentate hydrogen made by GLn20 and Glu405, could conform the struc tural explanation for the ability of most members of the family for di splaying both, glucosidase and galactosidase activity. (C) 1998 Academ ic Press Limited.