Crystallization and preliminary X-ray diffraction analysis of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp GK16

Glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp. GK16 prod uces glutaryl-7-aminocephalosporanic acid, a key intermediate for the synth esis of cephem antibiotics. Sequence alignment suggests that the enzyme may belong to the N-terminal nucleophile hydrolase superfamily including penic illin G acylase. The enzyme is an (alpha beta)(2) heterotetramer of two non identical subunits. These subunits are derived from a nascent precursor pol ypeptide that is cleaved proteolytically through a two-step autocatalytic p rocess upon folding. The enzyme has been crystallized using the vapor diffu sion method. A bipyramidal crystal form was obtained from a solution contai ning polyethylene glycol (MW 3350) and calcium chloride. Complete diffracti on data sets have been collected up to 2.8 Angstrom resolution. The crystal is tetragonal with the space group P4(1)2(1)2 or P4(3)2(1)2 and the unit c ell parameters are a = 5 = 73.5 Angstrom, c 380.3 Angstrom. Considerations of the possible values of V-m account for the presence of a tetramer in the asymmetric unit. (C) 2000 Academic Press.