TEM1 BETA-LACTAMASE STRUCTURE SOLVED BY MOLECULAR REPLACEMENT AND REFINED STRUCTURE OF THE S235A MUTANT

beta-Lactamases are bacterial enzymes which catalyse the hydrolysis of the beta-lactam ring of penicillins, cephalosporins and related compo unds, thus inactivating these antibiotics. The crystal structure of th e TEM1 beta-lactamase has been determined at 1.9 Angstrom resolution b y the molecular-replacement method, using the atomic coordinates of tw o homologous beta-lactamase refined structures which show about 36% st rict identity in their amino-acid sequences and 1.96 Angstrom r.m.s. d eviation between equivalent C alpha atoms. The TEM1 enzyme crystallize s in space group P2(1)2(1)2(1) and there is one molecule per asymmetri c unit. The structure was refined by simulated annealing to an R facto r of 15.6% for 15 086 reflections with I greater than or equal to 2 si gma(I) in the resolution range 5.0-1.9 Angstrom. The final crystallogr aphic structure contains 263 amino-acid residues, one sulfate anion in the catalytic cleft and 135 water molecules per asymmetric unit. The folding is very similar to that of the other known class A beta-lactam ases. It consists of two domains, the first is formed by a five-strand ed beta-sheet covered by three cr-helices on one face and one alpha-he lix on the other, the second domain contains mainly alpha-helices. The catalytic cleft is located at the interface between the two domains. We also report the crystallographic study of the TEM S235A mutant. Thi s mutation of an active-site residue specifically decreases the acylat ion rate of cephalosporins. This TEM S235A mutant crystallizes under t he same conditions as the wild-type protein and its structure was refi ned at 2.0 Angstrom resolution with an R value of 17.6%. The major mod ification is the appearance of a water molecule near the mutated resid ue, which is incompatible with the OG 235 present in the wild-type enz yme, and causes very small perturbations in the interaction network in the active site.