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

Martin A Day, David Jarrom, Andrew J Christofferson, Antonio E Graziano, J L Ross Anderson, Peter F Searle, Eva I Hyde, Scott A White

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Abstract

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.

Original languageEnglish
Pages (from-to)2601-2617
Number of pages17
JournalThe Biochemical journal
Volume478
Issue number13
Early online date18 Jun 2021
DOIs
Publication statusPublished - 16 Jul 2021

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© 2021 The Author(s).

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