Asymmetric ligand binding in homodimeric Enterobacter cloacae nitroreductase yields the Michaelis complex for nitroaromatic substrates.

Nitroreductase enzymes are of interest as antibiotic targets and as activators in enzyme prodrug therapy, but the precise substrate binding orientation and reaction mechanism are poorly understood. In order to design more effective antibiotics and improve enzyme prodrug therapy, an atomistic description of nitroaromatic substrate binding in the active Michaelis complex is highly desirable. Here, using an iterative molecular dynamics (MD) simulation protocol, the binding of p-nitrobenzoic acid (p-NBA) in oxidized and reduced Enterobacter cloacae nitroreductase (NR) was investigated. For the oxidized NR, the MD simulations distinguished between the two possible binding orientations of p-NBA in NR from X-ray crystal structure data. For the reduced NR, a distinct active binding orientation of p-NBA was found when the second active site of the NR homodimer was occupied by a NADH analogue. This model of the active Michaelis complex of p-NBA with NR provides a rationale for the reduction of p-NBA by NR via a hydride transfer reaction mechanism suggested by experimental results, and brings the proposed reaction mechanism from experiment and computational models into agreement.