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The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl–enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase. [ABSTRACT FROM AUTHOR]

Although commercialized inhibitors of active site serine β-lactamases are currently used in coadministration with antibiotic therapy, no clinically useful inhibitors of metallo-β-lactamases (MBLs) have yet been discovered. In this paper, we investigated the inhibitory effect of mercaptophosphonate derivatives against the three subclasses of MBLs (B1, B2, and B3). All 14 tested mercaptophosphonates, with the exception of 1a, behaved as competitive inhibitors for the three subclasses. Apart from 13and 21, all the mercaptophosphonates tested exhibit a good inhibitory effect on the subclass B2 MBL CphA with low inhibition constants (Ki< 15 μM). Interestingly, compound 18turned out to be a potent broad spectrum MBL inhibitor. The crystallographic structures of the CphA−10aand CphA−18complexes indicated that the sulfur atom of 10aand the phosphonato group of 18interact with the Zn2誉, respectively. Molecular modeling studies of the interactions between compounds 10aand 18and the VIM-4 (B1), CphA (B2), and FEZ-1 (B3) enzymes brought to light different binding modes depending on the enzyme and the inhibitor, consistent with the crystallographic structures. [ABSTRACT FROM AUTHOR]

The catalytic efficiency of the class D β-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of catalysis. Evidence is presented that the interaction between the indole group of Trp154 and the carboxylated lysine is essential for the stability of the posttranslationally modified Lys70. Substitution of Trp154 by Gly, Ala, or Phe yielded noncarboxylated enzymes which displayed poor catalytic efficiencies and reduced stability when compared to the wild-type OXA-10. The W154H mutant was partially carboxylated. In addition, the maximum values of kcat and kcat/KM were shifted toward pH 7, indicating that the carboxylation state of Lys70 is dependent on the protonation level of the histidine. A comparison of the three-dimensional structures of the different proteins also indicated that the Trp154 mutations did not modify the overall structures of OXA-10 but induced an increased flexibility of the 0-loop in the active site. Finally, the deacylation-impaired WI MA mutant was used to determine the structure of the acyl-enzyrne complex with benzylpenicillin. These results indicate a role of the Lys70 carboxylation during the deacylation step and emphasize the importance of Trp154 for the ideal positioning of active site residues leading to an optimum activity. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniaeML1708 exhibited a multiresistance phenotype, including resistance to all β-lactams tested, chloramphenicol, ciprofloxacin, nalidixic acid, tetracycline, and streptomycin. The double-disk synergy test was positive. ML1708 harbored a 50 kb conjugative plasmid that encoded a β-lactamase of pI 5.5. The corresponding blagene was identified by polymerase chain reaction and sequencing as a blaTEMgene. The deduced protein sequence revealed a new variant of TEM-1 β-lactamase designated TEM-164. TEM-164 contains the unusual following mutations: L40V and I279T. These modifications may result in a change of the pI to 5.5 and hydrolyze cefotaxime and ceftazidime. [ABSTRACT FROM AUTHOR]

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) β-lactamase from Aeromonas hydrophila is a Zn2+-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His118, Asp120, His196 and His263) and in substrate specificity (Val67, Thr157, Lys224 and Lys226), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp120 and His263, and thus is located in the ‘cysteine’ zinc-binding site. His118 and His196 residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val67 plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val67 also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys224 in the binding of substrate. Lys226 is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding. [ABSTRACT FROM AUTHOR]

The use of protein ESI mass spectrometry under non-denaturing conditions to analyze a dynamic combinatorial library of thiols/disulfides with the BcII metallo-β-lactamase enabled the rapid identification of an inhibitor with a Kiof <1 µM. The study exemplifies the utility of protein-MS for screening dynamic mixtures of potential enzyme–inhibitors. [ABSTRACT FROM AUTHOR]