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The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN

NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no stru...

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Autores principales: Day, Martin A., Jarrom, David, Christofferson, Andrew J., Graziano, Antonio E., Anderson, J. L. Ross, Searle, Peter F., Hyde, Eva I., White, Scott A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8286842/
https://www.ncbi.nlm.nih.gov/pubmed/34142705
http://dx.doi.org/10.1042/BCJ20210160
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author Day, Martin A.
Jarrom, David
Christofferson, Andrew J.
Graziano, Antonio E.
Anderson, J. L. Ross
Searle, Peter F.
Hyde, Eva I.
White, Scott A.
author_facet Day, Martin A.
Jarrom, David
Christofferson, Andrew J.
Graziano, Antonio E.
Anderson, J. L. Ross
Searle, Peter F.
Hyde, Eva I.
White, Scott A.
author_sort Day, Martin A.
collection PubMed
description NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH(−) to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor.
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spelling pubmed-82868422021-08-02 The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN Day, Martin A. Jarrom, David Christofferson, Andrew J. Graziano, Antonio E. Anderson, J. L. Ross Searle, Peter F. Hyde, Eva I. White, Scott A. Biochem J Enzymology NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH(−) to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor. Portland Press Ltd. 2021-07-16 2021-07-09 /pmc/articles/PMC8286842/ /pubmed/34142705 http://dx.doi.org/10.1042/BCJ20210160 Text en © 2021 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0/) . Open access for this article was enabled by the participation of University of Birmingham in an all-inclusive Read & Publish pilot with Portland Press and the Biochemical Society under a transformative agreement with JISC.
spellingShingle Enzymology
Day, Martin A.
Jarrom, David
Christofferson, Andrew J.
Graziano, Antonio E.
Anderson, J. L. Ross
Searle, Peter F.
Hyde, Eva I.
White, Scott A.
The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN
title The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN
title_full The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN
title_fullStr The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN
title_full_unstemmed The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN
title_short The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN
title_sort structures of e. coli nfsa bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to fmn
topic Enzymology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8286842/
https://www.ncbi.nlm.nih.gov/pubmed/34142705
http://dx.doi.org/10.1042/BCJ20210160
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