NDM-1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism

Abstract

The specter of a return to an era in which infectious disease looms as a significant threat to human health is not just hyperbole; there are serious concerns about the widespread overuse and misuse of antibiotics contributing to increased antibiotic resistance in pathogens. The recent discovery of a new enzyme, first identified in Klebsiella pneumoniae from a patient from New Delhi and denoted as NDM-1, represents an example of extreme promiscuity: It hydrolyzes and inactivates nearly all known β-lactam-based antibiotics with startling efficiency. NDM-1 can utilize different metal cofactors and seems to exploit an alternative mechanism based on the reaction conditions. Here we report the results of a combined experimental and theoretical study that examines the substrate, metal binding, and catalytic mechanism of the enzyme. We utilize structures obtained through X-ray crystallography, biochemical assays, and numerical simulation to construct a model of the enzyme catalytic pathway. The NDM-1 enzyme interacts with the substrate solely through zinc, or other metals, bound in the active site, explaining the observed lack of specificity against a broad range of β-lactam antibiotic agents. The zinc ions also serve to activate a water molecule that hydrolyzes the β-lactam ring through a proton shuttle.

Figure 1.

NDM-1 in complex with hydrolyzed ampicillin. Stereoview of the active site, with carbon atoms in the protein shown in cyan; those in the substrate are pink. Nitrogen atoms are blue, oxygen atoms are red, sulfur atoms are yellow, and zinc ions are magenta. An oriented water molecule (W) is gray. The F_o-F_c electron density around the substrate is contoured at 3σ. Dashed lines indicate key interactions between the protein and zinc ions.

Figure 2.

NDM-1 simulation active site. The protein is depicted as a surface and the (imipenem) substrate is colored as in Fig. 1, except for carbon atoms, which are cyan here. A) Substrate recognition is provided by interactions between the zinc ions and the carboxyl and carbonyl oxygen atoms of the substrate (dashed yellow lines) and nonspecific, generally hydrophobic interactions with the protein. The oriented water molecule occupies a pocket between the zinc ions. B) When the mobile loops move, a thin film of water surrounds the substrate, including 2 water molecules that serve as the nucleophile and as the source of the final proton required for ring cleavage (dashed green lines).

Figure 3.

Reaction mechanism. A) The oriented hydroxide ion serves as a general base to accept a proton from a bulk water molecule. B) A proton from the intermediate state is transferred to another bulk water molecule. C) The proton is then shuttled to the N4 nitrogen of the substrate. D) The nitrogen (N4)-carbon (C7) bond is cleaved in the final state. Energy values are estimates of the free energy from the QM/MM calculations. E) Comparison of the pathway energetics with a bulk water as the nucleophile (red, blue) or the oriented water acting as the nucleophile (green). Reaction coordinates correspond to steps shown in panels A–D.

Figure 4.

Dependence of NDM-1 turnover number (k_cat) on divalent cation concentration. The kinetic parameter was determined for NDM-1 using ampicillin as a substrate and increasing concentrations of Zn²⁺ (blue circles) or Cd²⁺ (red circles). Error bars are depicted in black.

Figure 5.

pH dependence of NDM-1 turnover number, k_cat. The kinetic parameter was determined for NDM-1 using CENTA as a substrate. Error bars are depicted in black.

Figure 6.

Structure of NDM-1 in complex with two cadmium ions and ampicillin. A) The intermediate (int-amp) is superposed with the modeled unhydrolyzed ampicillin (yellow). B) The hydrolyzed ampicillin (gray) from Δ30-amp-Zn is compared with the int-amp. Notice that the hydrolyzed ampicillin is rotated a few degrees (ccw) from the int-amp. C) The partially hydrolyzed ampicillin (green; intermediate) is fitted in the F_o-F_c electron density contoured at 2 σ level. The split Cd²⁺ is also indicated.