Abstract
Escherichia coli NfsB has been studied extensively for its potential for cancer gene therapy by reducing the prodrug CB1954 to a cytotoxic derivative. We have previously made several mutants with enhanced activity for the prodrug and characterised their activity in vitro and in vivo. Here, we determine the X-ray structure of our most active triple and double mutants to date, T41Q/N71S/F124T and T41L/N71S. The two mutant proteins have lower redox potentials than wild-type NfsB, and the mutations have lowered activity with NADH so that, in contrast to the wild-type enzyme, the reduction of the enzyme by NADH, rather than the reaction with CB1954, has a slower maximum rate. The structure of the triple mutant shows the interaction between Q41 and T124, explaining the synergy between these two mutations. Based on these structures, we selected mutants with even higher activity. The most active one contains T41Q/N71S/F124T/M127V, in which the additional M127V mutation enlarges a small channel to the active site. Molecular dynamics simulations show that the mutations or reduction of the FMN cofactors of the protein has little effect on its dynamics and that the largest backbone fluctuations occur at residues that flank the active site, contributing towards its broad substrate range.
| Original language | English |
|---|---|
| Article number | 5987 |
| Number of pages | 21 |
| Journal | International Journal of Molecular Sciences |
| Volume | 24 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - 22 Mar 2023 |
Bibliographical note
Funding Information:S.O.V. and M.A.D. were funded by Ph.D. studentships from the Medical Research Council (U.K).
Funding Information:
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). X-ray data collection and travel were supported by the European Synchrotron Radiation Facility via a Block Allocation Group scheme. We thank the beamline scientists for their help. Molecular graphics images were drawn using UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.
Publisher Copyright:
© 2023 by the authors.
