Xavier Raquet

X-ray analysis of the NMC-A beta-lactamase at 1.64-A resolution, a class A carbapenemase with broad substrate specificity

The treatment of infectious diseases by penicillin and cephalosporin antibiotics is continuously challenged by the emergence and the dissemination of the numerous TEM and SHV mutant β-lactamases with extended substrate profiles. These class A β-lactamases nevertheless remain inefficient against carbapenems, the most effective antibiotics against clinically relevant pathogens. A new member of this enzyme class, NMC-A, was recently reported to hydrolyze at high rates, and hence destroy, all known β-lactam antibiotics, including carbapenems and cephamycins. The crystal structure of NMC-A was solved to 1.64-Å resolution, and reveals modifications in the topology of the substrate-binding site. While preserving the geometry of the essential catalytic residues, the active site of the enzyme presents a disulfide bridge between residues 69 and 238, and certain other structural differences compared with the other β-lactamases. These unusual features in class A β-lactamases involve amino acids that participate in enzyme-substrate interactions, which suggested that these structural factors should be related to the very broad substrate specificity of this enzyme. The comparison of the NMC-A structure with those of other class A enzymes and enzyme-ligand complexes, indicated that the position of Asn-132 in NMC-A provides critical additional space in the region of the protein where the poorer substrates for class A β-lactamases, such as cephamycins and carbapenems, need to be accommodated.

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.

A disulfide bridge near the active site of carbapenem-hydrolyzing class A β-lactamases might explain their unusual substrate profile

Bacterial resistance to β‐lactam antibiotics, a clinically worrying and recurrent problem, is often due to the production of β‐lactamases, enzymes that efficiently hydrolyze the amide bond of the β‐lactam nucleus. Imipenem and other carbapenems escape the activity of most active site serine β‐lactamases and have therefore become very popular drugs for antibacterial chemotherapy in the hospital environment. Their usefulness is, however, threatened by the appearance of new β‐lactamases that efficiently hydrolyze them. This study is focused on the structure and properties of two recently described class A carbapenemases, produced by Serratia marcescens and Enterobacter cloacae strains and leads to a better understanding of the specificity of β‐lactamases. In turn, this will contribute to the design of better antibacterial drugs. Three‐dimensional models of the two class A carbapenemases were constructed by homology modeling. They suggested the presence, near the active site of the enzymes, of a disulfide bridge (C69‐C238) whose existence was experimentally confirmed. Kinetic parameters were measured with the purified Sme‐1 carbapenemase, and an attempt was made to explain its specific substrate profile by analyzing the structures of minimized Henri‐Michaelis complexes and comparing them to those obtained for the “classical” TEM‐1 β‐lactamase. The peculiar substrate profile of the carbapenemases appears to be strongly correlated with the presence of the disulfide bridge between C69 and C238. Proteins 27:47–58

Kinetic study of interaction between BRL 42715, beta-lactamases, and D-alanyl-D-alanine peptidases.

A detailed kinetic study of the interactions between BRL 42715, a beta-lactamase-inhibiting penem, and various beta-lactamases (EC 3.5.2.6) and D-alanyl-D-alanine peptidases (DD-peptidases, EC 3.4.16.4) is presented. The compound was a very efficient inactivator of all active-site serine beta-lactamases but was hydrolyzed by the class B, Zn(2+)-containing enzymes, with very different kcat values. Inactivation of the Streptomyces sp. strain R61 extracellular DD-peptidase was not observed, and the Actinomadura sp. strain R39 DD-peptidase exhibited a low level of sensitivity to the compound.

The rate‐limiting step in the folding of the cis‐Pro 167Thr mutant of TEM‐1 β‐lactamase is the trans to cis isomerization of a non‐proline peptide bond

The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM‐1 β‐lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166–Thr167 peptide bond, like the Glu166–Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild‐type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild‐type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans→cis isomerization of the Glu166–Thr167 peptide bond.

Thiolester substrates of DD-peptidases and beta-lactamases

With peptide substrates, the penicillin-sensitive dd-peptidases exhibit a strict specificity for d-Ala-d-Xaa C-termini. Only glycine is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes also hydrolyse various ester and thiolester analogues of their natural substrates. Some of the thiolesters whose C-terminal leaving group exhibited an l stereochemistry were significantly hydrolysed by some of the studied enzymes, particularly by the Actinomadura R39 dd-peptidase. By contrast, the strict specificity for a d residue in the penultimate position was fully retained. The same esters and thiolesters also behaved as substrates for β-lactamases. In this case, thiolesters exhibiting l stereochemistry in the C-terminal position could also be hydrolysed, mainly by the class C and class D enzymes. But, more surprisingly, the class C Enterobacter cloacae P99 β-lactamase also hydrolysed thiolesters containing an l residue in the penultimate position, sometimes more efficiently than the d isomer.