The 3D-structure, kinetics and dynamics of the E. coli nitroreductase NfsA with NADP+ provide glimpses of its catalytic mechanism.

Scott A White, Andrew J Christofferson, Alastair I Grainger, Martin A Day, David Jarrom, Antonio E Graziano, Peter F Searle, Eva I Hyde

FEBS letters 2022 Sep

filter terms:

Nitroreductases activate nitroaromatic antibiotics and cancer prodrugs to cytotoxic hydroxylamines and reduce quinones to quinols. Using steady-state and stopped-flow kinetics, we show that the Escherichia coli nitroreductase NfsA is 20-50 fold more active with NADPH than with NADH and that product release may be rate-limiting. The crystal structure of NfsA with NADP+ shows that a mobile loop forms a phosphate-binding pocket. The nicotinamide ring and nicotinamide ribose are mobile, as confirmed in molecular dynamics (MD) simulations. We present a model of NADPH bound to NfsA. Only one NADP+ is seen bound to the NfsA dimers, and MD simulations show that binding of a second NADP(H) cofactor is unfavourable, suggesting that NfsA and other members of this protein superfamily may have a half-of-sites mechanism. © 2022 Federation of European Biochemical Societies.

Citation

Scott A White, Andrew J Christofferson, Alastair I Grainger, Martin A Day, David Jarrom, Antonio E Graziano, Peter F Searle, Eva I Hyde. The 3D-structure, kinetics and dynamics of the E. coli nitroreductase NfsA with NADP+ provide glimpses of its catalytic mechanism. FEBS letters. 2022 Sep;596(18):2425-2440