Backbone assignment of E. coli NfsB and the effects of addition of the cofactor analogue nicotinic acid

Eva I Hyde; Alex Ka-Wing Chau; Lorna J Smith

doi:10.1007/s12104-020-09997-w

Backbone assignment of E. coli NfsB and the effects of addition of the cofactor analogue nicotinic acid

Eva I Hyde, Alex Ka-Wing Chau, Lorna J Smith

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Abstract

E. coli nitroreductase NfsB (also called NfnB) has been studied extensively, largely due to its potential for cancer gene therapy. A homodimeric flavoprotein of 216 residues, it catalyses the reduction of nitroaromatics to cytotoxic hydroxylamines by NADH and NADPH and also the reduction of quinones to hydroxyquinones. Its role in vivo is not known but it is postulated to be involved in reducing oxidative stress. The crystal structures of the wild type protein and several homologues have been determined in the absence and presence of ligands, including nicotinate as a mimic of the headpiece of the nicotinamide cofactors. There is little effect on the overall structure of the protein on binding ligands, but, from the B factors, there appears to be a decrease in mobility of 2 helices near the active site. As a first step towards examining the dynamics of the protein in solution with and without ligand, we have assigned the backbone 13C, 15N, and 1HN resonances of NfsB and examined the effect of the binding of nicotinate on the amide 15N, and 1HN shifts.

Original language	English
Pages (from-to)	143-151
Number of pages	9
Journal	Biomolecular NMR assignments
Volume	15
Issue number	1
Early online date	9 Jan 2021
DOIs	https://doi.org/10.1007/s12104-020-09997-w
Publication status	Published - 1 Apr 2021

Bibliographical note

Funding Information:
We thank Tom Frenkiel and Geoff Kelly, at the MRC Biomedical NMR centre at NIMR, London, for their assistance in collecting the triple resonance spectra at 800 MHz. We thank Gill Anlezark, (Centre for Applied Microbiology and Research, Porton Down, Salisbury UK) for the initial clone and samples of NfsB; Peter Searle (Department of Cancer Studies, University of Birmingham, UK) for cloning the nfsB gene into pET11c, Rosemary Parslow (School of Biosciences, University of Birmingham, UK) for skillful preparation of the protein and Jennifer Mitchell (Department of Chemistry, University of Oxford) for initial analysis of the NMR spectra while a student.

Funding Information:
This work was supported in part by the Medical Research Council, Grant Number G9815089. Spectra were collected at the NMR centre in Birmingham, funded by the Wellcome Trust and at the MRC Biomedical NMR centre at NIMR, London.

Publisher Copyright:
© 2021, The Author(s).

Keywords

Flavoprotein
Nicotinic acid
Nitroreductase
Titration

ASJC Scopus subject areas

Structural Biology
Biochemistry

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1007/s12104-020-09997-wLicence: Creative Commons: Attribution (CC BY)

Hyde_2021_Backbone_Assignment_Of_EColi_NfsBAFinal published version, 2.03 MBLicence: Creative Commons: Attribution (CC BY)

Structure - Function Studies of E. Coli Nitroreductase and Selected Mutants: A Route to Optimisation for Gene Therapy
Hyde, E. & Searle, P.
Medical Research Council
1/10/98 → 30/09/02
Project: Research Councils

Cite this

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abstract = "E. coli nitroreductase NfsB (also called NfnB) has been studied extensively, largely due to its potential for cancer gene therapy. A homodimeric flavoprotein of 216 residues, it catalyses the reduction of nitroaromatics to cytotoxic hydroxylamines by NADH and NADPH and also the reduction of quinones to hydroxyquinones. Its role in vivo is not known but it is postulated to be involved in reducing oxidative stress. The crystal structures of the wild type protein and several homologues have been determined in the absence and presence of ligands, including nicotinate as a mimic of the headpiece of the nicotinamide cofactors. There is little effect on the overall structure of the protein on binding ligands, but, from the B factors, there appears to be a decrease in mobility of 2 helices near the active site. As a first step towards examining the dynamics of the protein in solution with and without ligand, we have assigned the backbone 13C, 15N, and 1HN resonances of NfsB and examined the effect of the binding of nicotinate on the amide 15N, and 1HN shifts.",

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N2 - E. coli nitroreductase NfsB (also called NfnB) has been studied extensively, largely due to its potential for cancer gene therapy. A homodimeric flavoprotein of 216 residues, it catalyses the reduction of nitroaromatics to cytotoxic hydroxylamines by NADH and NADPH and also the reduction of quinones to hydroxyquinones. Its role in vivo is not known but it is postulated to be involved in reducing oxidative stress. The crystal structures of the wild type protein and several homologues have been determined in the absence and presence of ligands, including nicotinate as a mimic of the headpiece of the nicotinamide cofactors. There is little effect on the overall structure of the protein on binding ligands, but, from the B factors, there appears to be a decrease in mobility of 2 helices near the active site. As a first step towards examining the dynamics of the protein in solution with and without ligand, we have assigned the backbone 13C, 15N, and 1HN resonances of NfsB and examined the effect of the binding of nicotinate on the amide 15N, and 1HN shifts.

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Backbone assignment of E. coli NfsB and the effects of addition of the cofactor analogue nicotinic acid

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

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Projects

Structure - Function Studies of E. Coli Nitroreductase and Selected Mutants: A Route to Optimisation for Gene Therapy

Cite this