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 language | English |
|---|---|
| Pages (from-to) | 2601-2617 |
| Number of pages | 17 |
| Journal | Biochemical Journal |
| Volume | 478 |
| Issue number | 13 |
| Early online date | 18 Jun 2021 |
| DOIs | |
| Publication status | Published - 16 Jul 2021 |
Bibliographical note
Funding Information:M.A.D. was supported by an MRC PhD studentship; D.J. was supported by a BBSRC CASE studentship. X-ray data collection and travel was supported by the European Synchrotron Radiation Facility via a Block Allocation Group scheme. We thank the beamline scientists for their help. The MD simulations used the LIEF HPC-GPGPU Facility (resource grant pRMIT0007) hosted at the University of Melbourne, which was established with the assistance of LIEF Grant LE170100200. This MD research was also supported by computational resources provided by the Australian Government through the National Computational Infrastructure (NCI), under the National Computational Merit Allocation Scheme (project kl59 and resource grant uo96). Molecular graphics images were drawn using UCSF Chimera, developed by the Resource for Biocomputing, Visualisation and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.
Funding Information:
M.A.D. was supported by an MRC PhD studentship; D.J. was supported by a BBSRC CASE studentship. These are all the sources of direct funding for this project, other sources of income noire the fading are given in the acknowledgments.
Publisher Copyright:
© 2021 The Author(s).