Pre-Steady State Mechanistic Studies on NDM-1.

PALACIOS, A.R.; LLARRULL, L.I.; VILA, A.J.*

Gran Canaria, Islas Canarias

Workshop; 12th beta-lactamase Meeting (International Workshop); 2014

The emergence and dissemination of Gram-negative opportunistic bacterial pathogens that are resistant to all classes of β-lactam antibiotics is a public health issue of global concern. Metallo-β-Lactamases (MβLs) confer broad spectrum β-lactam resistance by using a Zn(II) center to catalyze the hydrolytic cleavage of the β-lactam ring, generating a product that lacks antimicrobial activity. Among them, NDM-1 is one of the most potent and widespread carbapenemases, raising an increasing clinical concern (1). NDM-1 belongs to subclass B1, which includes almost all plasmid-encoded MβLs. In this context, unveiling its catalytic mechanism is a prerequisite for the design of inhibitors or new β-lactam antibiotics. B1 enzymes can bind up to two Zn(II) ions in the active site, whose role in catalysis has been a matter of intense debate in the community (2). In this regard, the only reaction intermediates that have been trapped are anionic species on the nitrogen atom, that can only be stabilized by the Zn(II) ion at the Zn2 site (3). Here we report a mechanistic study by pre-steady state kinetics on substrate hydrolysis by NDM-1. First, we studied the kinetics of substrate binding to NDM-1 by monitoring quenching of the intrinsic Trp fluorescence upon binding (4). We then studied carbapenem hydrolysis by NDM-1 by means of a photodiode array. We found accumulation of a negatively charged reaction intermediate, which resembles the chemical features of those previously reported, suggesting a common reaction mechanism. This information is the cornerstone for the rational design of a common MβL inhibitor. References: 1. Mckenna M., Nature, 499:394-396, 2013; Cannon B., Nature, 509: S6-S8, 2014. 2. Badarau A., Page M.I., Biochemistry, 45(35):10654-66, 2006; Hawk M.J. et al., J. Am. Chem. Soc., 131(30), 10753-62, 2009; González J.M. et al., Nat. Chem. Biol., 8(8):698-700, 2012. 3. McManus-Munoz S. and Crowder M.W., Biochemistry., 38(5):1547-53, 1999; Wang Z., Fast W. and Benkovic S.J., Biochemistry, 38(31):10013-23, 1999; Tioni M.F. et al., J. Am. Chem. Soc. 130(47):15852-63, 2008. 4. Rasia R.M. and Vila A.J., J. Biol. Chem., 279(25):26046-51, 2004. Acknowledgements: ANPCyT, NHI, CONICET