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

DOI: 10.1002/1873-3468.14413

Authors: Scott A. White (University of Birmingham) , Andrew J. Christofferson (RMIT University) , Alastair I. Grainger (University of Birmingham; Aston University) , Martin A. Day (University of Birmingham) , David Jarrom (University of Birmingham) , Antonio E. Graziano (University of Birmingham) , Peter F. Searle (University of Birmingham) , Eva I. Hyde (University of Birmingham)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Febs Letters

State: Published (Approved)
Published: June 2022

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 E. 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.

Journal Keywords: Nitroreductase; molecular dynamics; flavoprotein; nitrofurazone; half-of-sites mechanism; NADP(H) binding; CB1954

Diamond Keywords: Bacteria; Enzymes

Subject Areas: Biology and Bio-materials, Medicine


Instruments: I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 05/06/2022 10:37

Discipline Tags:

Pathogens Antibiotic Resistance Infectious Diseases Health & Wellbeing Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)