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 prior computational mechanisms. Our mechanistic information provided a structural restraint for the orientation of bound substrate, placing the nitro group closer to the flavin N5 in the pocket that binds the amide of NADH. KIEs show that solvent provides a proton, enabling accommodation of different nitro group placements, consistent with NR’s broad repertoire.
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AFM and WP acknowledge invaluable discussions with D. Ballou and A. Kohen, and support from NSF I/UCRC grant 0969003 to the Center for Pharmaceutical Development. AFM recognizes WOCM for guidance, WP acknowledges the University of Kentucky for RCTF fellowship support. DR acknowledges support from N.I.H. grants NS38041 and GM110787 and N.S.F. grants MCB9904886. AFM and DR acknowledge support from N.S.F KY EPSCoR grant IIA-1355438.
Protein structures and data upon which they rest have been deposited to the Protein Data Bank under the accession codes 5J8D (Coordinates and structure factors for NAAD⋅NR) and 5J8G (Coordinates and structure factors for p-NBA⋅NR).
Supplemental Information includes seven figures and two tables and can be found with this article online at https://doi.org/10.1016/j.str.2017.05.002.
Pitsawong, Warintra; Haynes, Chad A.; Koder, Ronald L.; Rodgers, David W.; and Miller, Anne-Frances, "Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode for Catalytically Competent Nitroreductase" (2017). Chemistry Faculty Publications. 152.