New Delhi Metallo-β-Lactamase I: Substrate Binding and Catalytic Mechanism

Abstract

Metallo-β-lactamases can hydrolyze and deactivate lactam-containing antibiotics, which is the major mechanism for causing drug resistance in the treatment of bacterial infections. This has become a global concern because of the lack of clinically approved inhibitors so far. The emergence of New Delhi metallo-β-lactamase I (NDM-1) makes the situation even more serious. In this work, first, the structure of NDM-1 in complex with the inhibitor molecule l-captopril is investigated by both density functional theory (DFT) and hybrid quantum mechanical/molecular mechanical (QM/MM) methods, and the theoretical results are in good agreement with the X-ray structure. The Michaelis structure with an antibiotic compound (ampicillin) bound in the active site is constructed from a recent X-ray structure of the NDM-1 enzyme with hydrolyzed ampicillin. It is further simulated using a QM/MM molecular dynamics method. One of the interesting binding features of ampicillin in the NDM-1 active site is that the conserved C3 carboxylate group is not ligated with Zn2 but rather is only hydrogen-bonded with N220 and K211. A bridging hydroxide ion is suggested to connect two zinc cofactors. This hydroxide ion is also hydrogen-bonded with D124. Subsequent reaction path calculations indicate that the initial step of lactam ring-opening occurs through a concerted step in which the cleavage of the C-N bond and the transfer of the hydrogen bond to D124 are nearly concerted. The ligand bond between Zn2 and the C3 carboxylate group forms after the first step of nucleophilic addition. The calculated activation energy barrier height is about 19.4 kcal/mol for the hydrolysis of ampicillin, which can be compared with the experimental value of 15.8 kcal/mol derived from kcat = 15 s(-1). The overall mechanism is finally confirmed by a subsequent DFT study of a truncated active-site model.

Hydrolysis of cephalexin and meropenem by New Delhi metallo-β-lactamase: the substrate protonation mechanism is drug dependent

“…Development of inhibitors is majorly aided by the knowledge of structure and reactivity of the active site. Over the last few decades a number of crystallographic, 9-12 spectroscopic, [13][14][15][16][17][18][19][20][21][22][23][24] and computational 11,[25][26][27][28][29][30][31][32] studies have reported which shed light on the molecular structure and catalytic reactions of di-Zn metallo β−lactamases (MBLs), including NDM-1. Active site of NDM-1 has two Zn(II) binding sites bridged with a hydroxide (W1).…”

New Delhi metallo-β-lactamase (NDM)

“…RFQ-EXAFS studies showed that the Zn-Zn distance increased to 3.72 Å in a MBL L1-nitrocefin sample quenched at 10 ms. More recent structurefunction studies using cephalosporins containing p-substituted styrylbenzene substituents showed that the anionic nitrogen forms with other substrates and that the lifetime of the intermediate depends on the electron-donating ability of the styrylbenzene substituent 16 . Hammett analysis of these substrates supports the existence of two transient intermediates during catalysis: an oxyanion tetrahedral intermediate before C-N bond cleavage and an anionic nitrogen intermediate after hydrolysis 17 . Interestingly, the use of substrates lacking the styrylbenzene substituent does not result in the accumulation of the nitrogen anion intermediate, and C-N bond cleavage appears to be rate-limiting 18 .…”

Advances in the Experimental and Theoretical Understandings of Antibiotic Conjugated Gold Nanoparticles for Antibacterial Applications

“…Among them, S. aureus is one of the primary resistant pathogens which is found on the mucosal membrane and skin of around one‐third of the human population globally . Recently, NDM‐1 (New Delhi metallo‐beta‐lactamase‐1) producing K. pneumoniae strain was isolated from a Swedish patient of Indian origin from New Delhi, India . This carbapenem‐resistant Klebsiella strain was found to be resistant to all the antibiotics tested except colistin …”