Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data

Mechanistic and Spectroscopic Studies of Metallo-β-lactamase NDM-1

In an effort to biochemically characterize metallo-β-lactamase NDM-1, we cloned, overexpressed, purified, and characterized several maltose binding protein (MBP)–NDM-1 fusion proteins with different N-termini (full-length, Δ6, Δ21, and Δ36). All MBP–NDM-1 fusion proteins were soluble; however, only one, MBP–NDM-1Δ36, exhibited high activity and bound 2 equiv of Zn­(II). Thrombin cleavage of this fusion protein resulted in the truncated NDM-1Δ36 variant, which exhibited a <i>k</i>_cat of 16 s^–1 and a <i>K</i>_m of 1.1 μM when using nitrocefin as a substrate, bound 2 equiv of Zn­(II), and was monomeric in solution. Extended X-ray absorption fine structure studies of the NDM-1Δ36 variant indicate the average metal binding site for Zn­(II) in this variant consists of four N/O donors (two of which are histidines) and 0.5 sulfur donor per zinc, with a Zn–Zn distance of 3.38 Å. This metal binding site is very similar to those of other metallo-β-lactamases that belong to the B1 subclass. Pre-steady-state kinetic studies using nitrocefin and chromacef and the NDM-1Δ36 variant indicate that the enzyme utilizes a kinetic mechanism similar to that used by metallo-β-lactamases L1 and CcrA, in which a reactive nitrogen anion is stabilized and its protonation is rate-limiting. While they are very different in terms of amino acid sequence, these studies demonstrate that NDM-1 is structurally and mechanistically very similar to metallo-β-lactamase CcrA