The 3D-structure, Kinetics and Dynamics of the E. coli Nitroreductase NfsA with NADP Provide Glimpses of Its Catalytic Mechanism

Overview

Journal FEBS Lett

Specialty Biochemistry

Date 2022 Jun 1

PMID 35648111

Authors

Scott A White

Andrew J Christofferson

Alastair I Grainger

Martin A Day

David Jarrom

Antonio E Graziano

Peter F Searle

Eva I Hyde

Affiliations

Soon will be listed here.

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 superfamily may have a half-of-sites mechanism.

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