M. Vermeire

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 compounds, thus inactivating these antibiotics. The crystal structure of the TEM1 beta-lactamase has been determined at 1.9 A resolution by the molecular-replacement method, using the atomic coordinates of two homologous beta-lactamase refined structures which show about 36% strict identity in their amino-acid sequences and 1.96 A r.m.s. deviation between equivalent Calpha atoms. The TEM1 enzyme crystallizes in space group P2(1)2(1)2(1) and there is one molecule per asymmetric unit. The structure was refined by simulated annealing to an R-factor of 15.6% for 15 086 reflections with I >/= 2sigma(I) in the resolution range 5.0-1.9 A. The final crystallographic 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-lactamases. It consists of two domains, the first is formed by a five-stranded beta-sheet covered by three alpha-helices on one face and one alpha-helix 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. This mutation of an active-site residue specifically decreases the acylation rate of cephalosporins. This TEM S235A mutant crystallizes under the same conditions as the wild-type protein and its structure was refined at 2.0 A resolution with an R value of 17.6%. The major modification is the appearance of a water molecule near the mutated residue, which is incompatible with the OG 235 present in the wild-type enzyme, and causes very small perturbations in the interaction network in the active site.

The crystal structure of a penicilloyl-serine transferase of intermediate penicillin sensitivity. The DD-transpeptidase of streptomyces K15.

The serinedd-transpeptidase/penicillin-binding protein ofStreptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly. TheStreptomyces K15 enzyme is peculiar in that it can be considered as an intermediate between classical penicillin-binding proteins, for which benzylpenicillin is a very efficient inactivator, and the resistant penicillin-binding proteins that have a low penicillin affinity. With its moderate penicillin sensitivity, theStreptomyces K15 dd-transpeptidase would be helpful in the understanding of the structure-activity relationship of this penicillin-recognizing protein superfamily. The structure of theStreptomyces K15 enzyme has been determined by x-ray crystallography at 2.0-Å resolution and refined to anR-factor of 18.6%. The fold adopted by this 262-amino acid polypeptide generates a two-domain structure that is close to those of class A β-lactamases. However, the Streptomyces K15 enzyme has two particular structural features. It lacks the amino-terminal α-helix found in the other penicilloyl-serine transferases, and it exhibits, at its surface, an additional four-stranded β-sheet. These two characteristics might serve to anchor the enzyme in the plasma membrane. The overall topology of the catalytic pocket of the Streptomyces K15 enzyme is also comparable to that of the class A β-lactamases, except that the Ω-loop, which bears the essential catalytic Glu166residue in the class A β-lactamases, is entirely modified. This loop adopts a conformation similar to those found in theStreptomyces R61 dd-carboxypeptidase and class C β-lactamases, with no equivalent acidic residue.

The 4.5 Å resolution structure analysis of the exocellular DD-carboxypeptidase of Streptomyces albus G.

On the basis of their mechanistic properties, there exist two classess of DD-carboxypeptidases. The exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61 [l] and the membrane-bound DD-carboxypeptidases of several Bacilli [2,3], on the one hand, are serine-enzymes; they catalyse the attack of the sensitive amide bonds via the transitory formation of covalently serine-ester linked acylenzyme intermediates. The Streptomyces R61 enzyme has been crystallized [4]. The exocellular 18 000 M, DD-carboxy-peptidase of Streptomyces albus G (in short the G enzyme) [5], on the other hand, is probably a metallo (Zn2 ‘) enzyme [611, The apoprotein binds Zn2+ with K, -2 X 1014 M-r and this Zn2+ cofactor is required both for activity on substrate analogues (e.g., Ac2-L-Lys-D-Ala-DAla) and for binding of benzylpenicillin. The functioning of the active center, however, remains unknown. The G enzyme has been crystallized [7] into well-formed prismatic crystals (space group P2,; unit cell dimensions CI = 5 1 .l A, b = 49.7 A, c = 38.7 A, 0 = 100.6 A; 2 enzyme molecules/unit cell). This paper presents the 4.5 A resolution structure analysis of the G enzyme and studies which have permitted visualization of the enzyme active center and localization of the Zn2 + binding site.

Crystal structure of Enterobacter cloacae 908R class C beta-lactamase bound to iodo-acetamido-phenyl boronic acid, a transition-state analogue.

The structures of the class C β-lactamase from Enterobacter cloacae 908R alone and in complex with a boronic acid transition-state analogue were determined by X-ray crystallography at 2.1 and 2.3 Å, respectively. The structure of the enzyme resembles those of other class C β-lactamases. The structure of the complex with the transition-state analogue, iodo-acetamido-phenyl boronic acid, shows that the inhibitor is covalently bound to the active-site serine (Ser64). Binding of the inhibitor within the active site is compared with previously determined structures of complexes with other class C enzymes. The structure of the boronic acid adduct indicates ways to improve the affinity of this class of inhibitors. This structure of 908R class C β-lactamase in complex with a transition-state analogue provides further insights into the mechanism of action of these hydrolases.