The flavoenzyme NTR (nitroreductase nfsB from Escherichia coli) and the prodrug CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) have been found to be a good potential combination for virus-directed enzyme prodrug therapy (VDEPT). However, wild-type NTR has poor kinetics and binding with CB1954, and the mechanism for the reduction of CB1954 by NTR is poorly understood. The aim of this work has been to investigate, using quantum mechanical computational methods, the potential underlying reaction mechanisms so as to identify the order of electron and proton transfers, and source of the protons, that make up the initial reduction step. Additionally, molecular mechanics and molecular dynamics have been used to examine the nature of the active site of the wild-type enzyme, as well as several mutants, and determine possible binding modes of the substrate. Finally, ONIOM calculations were utilised to examine substrate orientations and electronic states at a quantum mechanical level with key active site amino acids present at a molecular mechanics level. Calculations with the MPW1PW91 density functional and 6-31G** basis set yielded a single gas phase transition state geometry for the hydride transfer from the FMN (flavin mononucleotide) cofactor of NTR to a nitro group of CB1954. Additionally, three reaction profiles were generated which suggest that in the gas phase, the reduction proceeds by electron transfer from FMN, proton transfer , then second proton transfer and electron transfer concerted with N-O bond breaking, regardless of the source (FMN or solution) or order of the protons. Molecular mechanics calculations with the FF03 force field found a binding mode with the amide group of CB1954 bound in the active site of NTR—an orientation ideally suited for an electron transfer mechanism
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