Characterization of purified New Delhi metallo-β-lactamase-1.
New Delhi metallo-β-lactmase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to almost all clinically used β-lactam antibiotics, its presence within an easily transmissible plasmid bearing a number of other antibiotic resistance determinants, its carriage in a variety of enterobacteria, and its presence in both nosocomial and community-acquired infections. To improve our understanding of the molecular basis of this threat, NDM-1 was purified and characterized. Recombinant NDM-1 bearing its native leader sequence was expressed in Escherichia coli BL21 cells. The major processed form found to be released into culture media contains a 35-residue truncation at the N-terminus. This form of NDM-1 is monomeric and can be purified with 1.8 or 1.0 equiv of zinc ion, depending on the experimental conditions. Treatment of dizinc NDM-1 with EDTA results in complete removal of both zinc ions, but the relatively weaker chelator PAR chelates only 1 equiv of zinc ion from folded protein but 1.9 equiv of zinc ion from denatured protein, indicating different affinities for each metal binding site. UV-vis spectroscopy of the dicobalt metalloform along with molecular dynamics simulations of the dizinc metallo form indicates that the dinuclear metal cluster at the active site of NDM-1 is similar in structure to other class B1 metallo-β-lactamases. Supplementation of excess zinc ions to monozinc NDM-1 has differential effects on enzyme activity with respect to three different classes of β-lactam substrates tested, penems, cephems, and carbapenems, and likely reflects dissimilar contributions of the second equivalent of metal ion to the catalysis of the hydrolysis of these substrates. Fits to these concentration dependencies are used to approximate the K(d) value of the more weakly bound zinc ion (2 μM). NDM-1 achieved maximal activity with all substrates tested when supplemented with approximately 10 μM ZnSO(4), displaying k(cat)/K(M) values ranging from 1.4 × 10(6) to 2.0 × 10(7) M(-1) s(-1), and a slight preference for cephem substrates. This work provides a foundation for an improved understanding of the molecular basis of NDM-1-mediated antibiotic resistance and should allow more quantitative studies to develop targeted therapeutics.
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712, USA.
Pei W
PW
Thomas
Min
M
Zheng
Shanshan
S
Wu
Hua
H
Guo
Dali
D
Liu
Dingguo
D
Xu
Walter
W
Fast
D003035:Cobalt / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004927:Escherichia coli Infections / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D006868:Hydrolysis; D007710:Klebsiella Infections / Q000201:enzymology; D007711:Klebsiella pneumoniae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D008670:Metals; D011994:Recombinant Proteins; D003035:Cobalt; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1; D015032:Zinc