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

ABSTRACT: 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 superfa

SUBMITTER: White SA 

PROVIDER: S-EPMC9912195 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature