The problem of a solvent exposable disulfide when preparing Co(II)-substituted metallo-β-lactamase L1 from Stenotrophomonas maltophilia

Crowder, Michael W.; Yang, Ke‐Wu; Carenbauer, Anne L.; Periyannan, Gopal R.; Seifert, Mary E.; Rude, Nathan E.; Walsh, Timothy R.

doi:10.1007/s007750000173

Cited by 27 publications

(42 citation statements)

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“…Co(II)-substitution has been largely exploited as a fruitful strategy to obtain structural information about the active sites of zinc enzymes by employing the spectroscopic features of Co(II) (38). This characterization has been also applied to MBLs (16,19,39,40). Despite the fact that the four point mutations found in M5 did not occur in the active site, we prepared the di-Co(II) derivative of this mutant to check whether these mutations could have some effect on the metal site structure.…”

Section: Resultsmentioning

confidence: 99%

Mimicking natural evolution in metallo-β-lactamases through second-shell ligand mutations

Tomatis

Rasia

Segovia

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Metallo-␤-lactamases (MBLs) represent the latest generation of ␤-lactamases. The structural diversity and broad substrate profile of MBLs allow them to confer resistance to most ␤-lactam antibiotics. To explore the evolutionary potential of these enzymes, we have subjected the Bacillus cereus MBL (BcII) to a directed evolution scheme, which resulted in an increased hydrolytic efficiency toward cephalexin. A systematic study of the hydrolytic profile, substrate binding, and active-site features of the evolved lactamase reveal that directed evolution has shaped the active site by means of remote mutations to better hydrolyze cephalosporins with small, uncharged C-3 substituents. One of these mutations is found in related enzymes from pathogenic bacteria and is responsible for the increase in that enzyme's hydrolytic profile. The mutations lowered the activation energy of the rate-limiting step rather than improved the affinity of the enzyme toward these substrates. The following conclusions can be made: (i) MBLs are able to expand their substrate spectrum without sacrificing their inherent hydrolytic capabilities; (ii) directed evolution is able to mimic mutations that occur in nature; (iii) the metal-ligand strength is tuned by second-shell mutations, thereby influencing the catalytic efficiency; and (iv) changes in the position of the second Zn(II) ion in MBLs affect the substrate positioning in the active site. Overall, these results show that the evolution of enzymatic catalysis can take place by remote mutations controlling reactivity.Co(II)-substitution ͉ directed evolution ͉ zinc enzymes ͉ antibiotic resistance T he ␤-lactam antibiotics account for more than half of the world's antibiotic market and are one of the cornerstones of antibacterial chemotherapy (1). The increasing use of these drugs, both in the clinical setting and in animal feed, induces the development of different resistance mechanisms in pathogenic and opportunistic microorganisms (2, 3). Among them, the expression of ␤-lactamases is predominant. In the last decade, there has been growing concern about the dissemination of genes coding for metallo-␤-lactamases (MBLs) among infectious microorganisms (4-7). MBLs are zinc-dependent enzymes that hydrolyze ␤-lactams by favoring the deprotonation of a metalbound water molecule, which (as a hydroxide) is a powerful nucleophile (8-11). Much recent literature has been devoted to the elucidation of the structure and mechanism of action of . Parallel to these efforts, the significant growth of new MBL sequences has revealed an initially unforeseen molecular and structural diversity in this family of enzymes (6). This diversity encompasses changes in the coordination features of the Zn(II) ions as well as in the topology of the active site. This fact has hitherto thwarted the successful development of clinically useful inhibitors. The potential danger of MBLs is further enhanced by the broad substrate spectrum displayed by these enzymes, as compared with the better-characterized Serdependent ␤-lactamas...

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Section: Resultsmentioning

confidence: 99%

Mimicking natural evolution in metallo-β-lactamases through second-shell ligand mutations

Tomatis

Rasia

Segovia

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…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.…”

Section: Methodsmentioning

confidence: 99%

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

Garrity

Carenbauer

Herron

et al. 2004

Journal of Biological Chemistry

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Metallo-␤-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis. The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a ␤-lactamase fold. In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and presteady-state and steady-state kinetics. The D120C, D120N, and D120S mutants were shown to bind 1.6 ؎ 0.2, 1.8 ؎ 0.2, and 1.1 ؎ 0.2 mol of Zn(II) per monomer, respectively. The mutants exhibited 10-to 1000-fold drops in k cat values as compared with wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested. Solvent isotope and pH dependence studies indicate one or more protons in flight, with pK a values outside the range of pH 5-10 (except D120N), during a rate-limiting step for all the enzymes. These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme. This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction.

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“…2B). The intensity of this peak implies that it is due to a ligand field transition rather than from a ligand to metal charge transfer band (expected ⑀ of Ͼ500 M Ϫ1 cm Ϫ1 ) (40). The position of this peak suggests that it arises from Co(III) in the sample, indicating that Co(II) is air-oxidizing to Co(III) during protein preparation at pH 7.5.…”

Section: Preparation and Characterization Of Co(ii)-substitutedmentioning

confidence: 95%

A Five-coordinate Metal Center in Co(II)-substituted VanX

Breece

Costello

Bennett

et al. 2005

Journal of Biological Chemistry

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The problem of a solvent exposable disulfide when preparing Co(II)-substituted metallo-β-lactamase L1 from Stenotrophomonas maltophilia

Cited by 27 publications

References 0 publications

Mimicking natural evolution in metallo-β-lactamases through second-shell ligand mutations

Mimicking natural evolution in metallo-β-lactamases through second-shell ligand mutations

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

A Five-coordinate Metal Center in Co(II)-substituted VanX

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