Biochemical, Mechanistic, and Spectroscopic Characterization of Metallo-β-lactamase VIM-2

Journal Article · Tue Nov 25 00:00:00 EST 2014 · Biochemistry

DOI:https://doi.org/10.1021/bi500916y· OSTI ID:1228935

Aitha, Mahesh ^[1]; Marts, Amy R. ^[1]; Bergstrom, Alex ^[1]; Møller, Abraham Jon ^[1]; Moritz, Lindsay ^[1]; Turner, Lucien ^[1]; Nix, Jay C. ^[2]; Bonomo, Robert A. ^[3]; Page, Richard C. ^[1]; Tierney, David L. ^[1]; Crowder, Michael W. ^[1]

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1. Miami Univ., Oxford, OH (United States)
2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
3. Louis Stokes Cleveland Dept. of Veterans Affairs Medical Center, Cleveland, OH (United States); Case Western Reserve Univ., Cleveland, OH (United States)

Our study examines metal binding to metallo-β-lactamase VIM-2, demonstrating the first successful preparation of a Co(II)-substituted VIM-2 analogue. Spectroscopic studies of the half- and fully metal loaded enzymes show that both Zn(II) and Co(II) bind cooperatively, where the major species present, regardless of stoichiometry, are apo- and di-Zn (or di-Co) enzymes. We also determined the di-Zn VIM-2 structure to a resolution of 1.55 Å, and this structure supports results from spectroscopic studies. Kinetics, both steady-state and pre-steady-state, show that VIM-2 utilizes a mechanism that proceeds through a very short-lived anionic intermediate when chromacef is used as the substrate. Comparison with other B1 enzymes shows that those that bind Zn(II) cooperatively are better poised to protonate the intermediate on its formation, compared to those that bind Zn(II) non-cooperatively, which uniformly build up substantial amounts of the intermediate.

• Research Organization: Brookhaven National Lab. (BNL), Upton, NY (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Basic Energy Sciences (BES)
• DOE Contract Number: SC00112704
• OSTI ID: 1228935
• Report Number(s): BNL-111010-2015-JA
• Journal Information: Biochemistry, Vol. 53, Issue 46; ISSN 0006-2960
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES