X-ray structure of the Asn276Asp variant of the Escherichia coli TEM-1 beta-lactamase: direct observation of electrostatic modulation in resistance to inactivation by clavulanic acid.

The clinical use of beta-lactam antibiotics combined with beta-lactamase inactivators, such as clavulanate, has resulted in selection of beta-lactamases that are insensitive to inactivation by these molecules. Therefore, therapeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme. The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 variants are designated as inhibitor-resistant TEM ("IRT") enzymes. Three mutations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution. In this study, we have characterized the kinetic properties of the inhibition process of the wild-type TEM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam, and we report the X-ray structure for the mutant variant at 2.3 A resolution. The changes in kinetic parameters for the interactions of the inhibitors with the wild-type and the mutant enzymes were more pronounced for clavulanate, and relatively inconsequential for sulbactam and tazobactam. The structure of the Asn276Asp mutant enzyme revealed a significant movement of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate. A water molecule critical for the inactivation chemistry by clavulanate, which is observed in the wild-type enzyme structure, is not present in the crystal structure of the mutant variant. Such structural changes favor the turnover process over the inactivation chemistry for clavulanate, with profound phenotypic consequences. The report herein represents the best studied example of inhibitor-resistant beta-lactamases.

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 beta-lactamases with extended substrate profiles. These class A beta-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 beta-lactam antibiotics, including carbapenems and cephamycins. The crystal structure of NMC-A was solved to 1.64-A 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 beta-lactamases. These unusual features in class A beta-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 beta-lactamases, such as cephamycins and carbapenems, need to be accommodated.

Structural basis of extended spectrum TEM beta-lactamases. Crystallographic, kinetic, and mass spectrometric investigations of enzyme mutants.

The E166Y and the E166Y/R164S TEM-1 beta-lactamase mutant enzymes display extended spectrum substrate specificities. Electrospray mass spectrometry demonstrates that, with penicillin G as substrate, the rate-limiting step in catalysis is the hydrolysis of the E166Y acyl-enzyme complex. Comparison of the 1.8-A resolution x-ray structures of the wild-type and of the E166Y mutant enzymes shows that the binding of cephalosporin substrates is improved, in the mutant enzyme, by the enlargement of the substrate binding site. This enlargement is due to the rigid body displacement of 60 residues driven by the movement of the omega-loop. These structural observations strongly suggest that the link between the position of the omega-loop and that of helix H5, plays a central role in the structural events leading to extended spectrum TEM-related enzymes. The increased omega-loop flexibility caused by the R164S mutation, which is found in several natural mutant TEM enzymes, may lead to similar structural effects. Comparisons of the kinetic data of the E166Y, E166Y/R164S, and R164S mutant enzymes supports this hypothesis.

Is the function of the cdc2 kinase subunit proteins tuned by their propensities to oligomerize? Conformational states in solution of the cdc2 kinase partners p13suc1 and p9cksphy.

The cdc2 kinase subunit (cks) proteins play an essential function in the control of mitosis through their molecular complexes with the cdc2 kinase. In this work, we characterize the conformational state(s) in solution of the cks proteins p13suc1 from Schizosaccharomyces pombe and p9cksphy from Physarum polycephalum. Monomers of p13suc1 and p9cksphy were found to be markedly nonglobular, presumably with a long, nonfolded C-terminal moiety. This was in contrast to the previously published structure of p13suc1, derived from crystallographic studies on a zinc-promoted p13suc1 dimer, in which the individual p13suc1 subunits had a globular conformation. This disparity was resolved when we found that the globular p13suc1 fold undergoes a conformational transition into nonglobular monomers upon dissociation of the dimers following chelation of the zinc ions by ethylenediaminetetraacetic acid (EDTA). We also found that p13suc1, but not p9cksphy, forms stable dimers in the absence of metal ions. The topology of these EDTA-insensitive dimers likely resembles that of the human p9ckshs2 protein, characterized by beta 4 strand exchange from each nonglobular monomer.

The asparagine to aspartic acid substitution at position 276 of TEM-35 and TEM-36 is involved in the beta-lactamase resistance to clavulanic acid.

TEM-35 (inhibitor resistant TEM (IRT)-4) and TEM-36 (IRT-7) clavulanic acid-resistant beta-lactamases have evolved from TEM-1 beta-lactamase by two substitutions: a methionine to a leucine or a valine at position 69 and an asparagine to an aspartic acid at position 276. The substitutions at position 69 have previously been shown to be responsible for the resistance to clavulanic acid, and they are the only mutations encountered in TEM-33 (IRT-5) and TEM-34 (IRT-6). However, the N276D substitution has never been found alone in inhibitor-resistant beta-lactamases, and its role in resistance to clavulanic acid was thus unclear. The N276D mutant was constructed, purified, and kinetically characterized. It was shown that the substitution has a direct effect on substrate affinities and leads to slightly decreased catalytic efficiencies and that clavulanic acid becomes a poor substrate of the enzyme. Electrospray mass spectrometry demonstrated the simultaneous presence of free and inhibited enzymes after incubation with clavulanic acid and showed that a cleaved moiety of clavulanic acid leads to the formation of the major inactive complex. The kinetic properties of the N276D mutant could be linked to a salt-bridge interaction of aspartic acid 276 with arginine 244 that alters the electrostatic properties in the substrate binding area.

Electrostatic analysis of TEM1 beta-lactamase: effect of substrate binding, steep potential gradients and consequences of site-directed mutations.

BACKGROUND: Escherichia coli TEM1 is a penicillinase and belongs to class A beta-lactamases. Its naturally occurring mutants are responsible for bacterial resistance to beta-lactamin-based antibiotics. X-ray structure determinations show that all class A beta-lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated. We address the questions of what the molecular contexts during the acylation and deacylation steps are and how they contribute to the efficiency of these penicillinases. RESULTS: Electrostatic analysis of the 1.8 A resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl-enzyme complexes, showed that substrate binding induces an upward shift in the pKa of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote acylation. In the acyl-enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the estercarbonyl carbon of the acyl-enzyme complex, in an electrostatic potential gradient amounting to 30 kTe-1 A-1. Other residues, not directly involved in catalysis, also contribute to the formation of this gradient. The deacylation rate is related to the magnitude of the gradient. The kinetic behavior of site-directed mutants that affect the protonation state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism. CONCLUSIONS: In the wild-type enzyme, the very high rates of acylation and deacylation of class A beta-lactamases arise from an optimal chemical setup in which the acylation reaction seems triggered by substrate binding that changes the general base property of Lys73. In site-directed mutants where Lys73 is protonated, acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.