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.

QM/MM studies of the hydrolysis of a β-lactam antibiotic molecule (biapenem) catalyzed by a mono-zinc β-lactamase (CphA) reveal the complete reaction mechanism and show that an experimentally determined enzyme-intermediate complex is a stable intermediate or product in a minor pathway.

Angiotensin-converting enzyme (ACE) is an important zinc-dependent hydrolase responsible for converting the inactive angiotensin I to the vasoconstrictor angiotensin II and for inactivating the vasodilator bradykinin. However, the substrate binding mode of ACE has not been completely understood. In this work, we propose a model for an ACE Michaelis complex based on two known X-ray structures of inhibitor-enzyme complexes. Specifically, the human testis angiotensin-converting enzyme (tACE) complexed with two clinic drugs were first investigated using a combined quantum mechanical and molecular mechanical (QM/MM) approach. The structural parameters obtained from the 550 ps molecular dynamics simulations are in excellent agreement with the X-ray structures, validating the QM/MM approach. Based on these structures, a model for the Michaelis complex was proposed and simulated using the same computational protocol. Implications to ACE catalysis are discussed.

The angiotensin-converting enzyme (ACE) exhibits critical functions in the conversion of angiotensin I to angiotensin II and the degradation of bradykinin and other vasoactive peptides. As a result, the ACE inhibition has become a promising approach in the treatment of hypertension, heart failure, and diabetic nephropathy. Extending our recent molecular dynamics simulation of the testis ACE in complex with a bona fide substrate molecule, hippuryl-histidyl-leucine, we presented here a detailed investigation of the hydrolytic process and possible influences of the chloride ion on the reaction using a combined quantum mechanical and molecule mechanical method. Similar to carboxypeptidase A and thermolysin, the promoted water mechanism is established for the catalysis of ACE. The E384 residue was found to have the dual function of a general base for activating the water nucleophile and a general acid for facilitating the cleavage of amide C-N bond. Consistent with experimental observations, the chloride ion at the second binding position is found to accelerate the reaction rate presumably due to the long-range electrostatic interactions but has little influence on the overall substrate binding characteristics.