Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode for Catalytically Competent Nitroreductase.

ABSTRACT: Nitroreductase (NR) from Enterobacter cloacae 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 prior computational mechanisms. Our mechanistic information provided a struct

SUBMITTER: Pitsawong W 

PROVIDER: S-EPMC5560098 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature