Physical Sciences and Mathematics Commons^™

Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode For Catalytically Competent Nitroreductase, Warintra Pitsawong, Chad A. Haynes, Ronald L. Koder, David W. Rodgers, Anne-Frances Miller

Chemistry Faculty Publications

Nitroreductase from Enterobacter cloacae (NR) reduces diverse nitroaromatics including herbicides, explosives and prodrugs, and holds promise for bioremediation, prodrug activation and enzyme-assisted synthesis. We solved crystal structures of NR complexes with bound substrate or analog for each of its two half-reactions. We complemented these with kinetic isotope effect (KIE) measurements elucidating H-transfer steps essential to each half-reaction. KIEs indicate hydride transfer from NADH to the flavin consistent with our structure of NR with the NADH analog nicotinic acid adenine dinucleotide (NAAD). The KIE on reduction of p-nitrobenzoic acid (p-NBA) also indicates hydride transfer, and requires revision of …