title: Mechanistic and spectroscopic studies of metallo-β-lactamase NDM-1.
abstract: 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 k(cat) of 16 s(-1) and a K(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.
authors.affiliation: Department of Chemistry and Biochemistry, 160 Hughes Hall, Miami University, Oxford, Ohio 45056, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Yang
authors.forename: Mahesh
authors.initials: M
authors.lastname: Aitha
authors.forename: Alyssa M
authors.initials: AM
authors.lastname: Hetrick
authors.forename: Timothy K
authors.initials: TK
authors.lastname: Richmond
authors.forename: David L
authors.initials: DL
authors.lastname: Tierney
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain; D002511:Cephalosporins / Q000378:metabolism; D007700:Kinetics; D008027:Light; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012542:Scattering, Radiation; D013917:Thrombin / Q000378:metabolism; D056928:X-Ray Absorption Spectroscopy; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002511:Cephalosporins; D011993:Recombinant Fusion Proteins; D013917:Thrombin; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1; C063912:carbapenemase; C021720:nitrocefin; D015032:Zinc