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

In an effort to biochemically characterize metallo-beta-lactamase NDM-1, we cloned, overexpressed, purified, and characterized several maltose binding protein (MBP)-NDM-1 fusion proteins with different N-termini (full-length, Delta 6, Delta 21, and Delta 36). All MBP-NDM-1 fusion proteins were soluble; however, only one, MBP NDM-1 Delta 36, exhibited high activity and bound 2 equiv of Zn(II). Thrombin cleavage of this fusion protein resulted in the truncated NDM-1 Delta 36 variant, which exhibited a k(cat) of 16 s(-1) and a K-m of 1.1 mu M when using nitrocefin as a substrate, bound 2 equiv of Zn(11), and was monomeric in solution. Extended X-ray absorption fine structure studies of the NDM-1 Delta 36 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 angstrom. This metal binding site is very similar to those of other metallo-beta-lactamases that belong to the B1 subclass. Pre-steady-state kinetic studies using nitrocefin and chromacef and the NDM-1 Delta 36 variant indicate that the enzyme utilizes a kinetic mechanism similar to that used by metallo-beta-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-beta-lactamase CcrA.