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Abstract

BackgroundThe metallo-β-lactamases are Zn(II)-containing enzymes that hydrolyze the β-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-β-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-β-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics.ResultsSite-directed mutations were generated of amino acids previously predicted to be important in substrate binding. Steady-state kinetic studies using the mutant enzymes and 9 different substrates demonstrated varying Km and kcat values for the different enzymes and substrates and that no direct correlation between Km and the effect of the mutation on substrate binding could be drawn. Stopped-flow fluorescence studies using nitrocefin as the substrate showed that only the S224D and Y228A mutants exhibited weaker nitrocefin binding.ConclusionsThe data presented herein indicate that Ser224, Ile164, Phe158, Tyr228, and Asn233 are not essential for tight binding of substrate to metallo-β-lactamase L1. The results in this work also show that Km values are not reliable for showing substrate binding, and there is no correlation between substrate binding and the amount of reaction intermediate formed during the reaction. This work represents the first experimental testing of one of the computational models of the metallo-β-lactamases.

Probing the Specificity of the Subclass B3 FEZ-1 Metallo-β-lactamase by Site-directed Mutagenesis

“…The crystal structure of the L1 ␤-lactamase shows that there is a large loop that extends over the active site, and modeling studies have predicted that Phe 156 (for L1) and Tyr 156 (for FEZ-1) can make significant contacts with large substituents at the C-6 or C-7 positions in penicillins or cephalosporins, respectively. In L1, the F156A mutation yields a ␤-lactamase with a catalytic efficiency similar to that of the wild type (32). In consequence, the experimental data do not support the conclusions of the modeling studies (18).…”

Metal Binding Asp-120 in Metallo-β-lactamase L1 from Stenotrophomonas maltophilia Plays a Crucial Role in Catalysis

“…Mutations were introduced into the L1 gene by using the overlap extension method of Ho et al (47), as described previously (48,49). The oligonucleotides used for the preparation of the mutants are as follows: D120C forward, CACgCACACgCCTgCCATgCCggACCggTg; D120C reverse, CACCggTCCggCATggCAggCgTgTgCgTg; D120N forward, CACgCACACgCCAACCATgCCggACCggTg; D120N reverse, CACCggTCCggCATggTTggCgTgTgCgTg; D120S forward, CACgCACACgCCAgCCATgCCggACCggTg; and D120S reverse, CACCggTCCggCATggCTggCgTgTgCgTg.…”

“…After confirmation of the sequence, the mutated pL1pUC19 plasmid was digested with NdeI and HindIII, and the 900-bp, mutated L1 gene was gel-purified and ligated into pET26b to create the mutant overexpression plasmids. To test for overexpression of the mutant enzymes, E. coli BL21(DE3)pLysS cells were transformed with the mutated overexpression plasmids, and small scale growth cultures were used (48). Large-scale (4 liters) preparations of the L1 mutants were performed as described previously (36).…”

Probing the Dynamics of a Mobile Loop above the Active Site of L1, a Metallo-β-lactamase from Stenotrophomonas maltophilia, via Site-directed Mutagenesis and Stopped-flow Fluorescence Spectroscopy

“…The DNA sequences were analyzed using a PerkinElmer Life Sciences ABI 5300 Genetic Analyzer. To test for overexpression of the mutant enzymes, E. coli BL21(DE3)pLysS cells were transformed with the mutated overexpression plasmids, and small scale growth cultures were used (45). Large scale (4-liter) preparations of the L1 mutants were performed as described previously (34), with the exception of W53F and W206F.…”

“…Circular dichroism samples were prepared by dialyzing the purified enzyme samples versus 3ϫ 2 liters of 5 mM phosphate buffer, pH 7.0, over 6 h. The samples were diluted with final dialysis buffer to ϳ75 g/ml. A Jasco J-810 CD spectropolarimeter operating at 25°C was used to collect CD spectra (34,45).…”