8C5E
E. coli NfsB-T41Q/N71S/F124T mutant bound to nicotinic acid
Summary for 8C5E
| Entry DOI | 10.2210/pdb8c5e/pdb |
| Descriptor | Oxygen-insensitive NAD(P)H nitroreductase, FLAVIN MONONUCLEOTIDE, NICOTINIC ACID, ... (5 entities in total) |
| Functional Keywords | nitroreductase mutant, complex, oxidoreductase |
| Biological source | Escherichia coli |
| Total number of polymer chains | 2 |
| Total formula weight | 48740.81 |
| Authors | White, S.A.,Hyde, E.I.,Day, M.A. (deposition date: 2023-01-06, release date: 2023-04-19, Last modification date: 2024-06-19) |
| Primary citation | Day, M.A.,Christofferson, A.J.,Anderson, J.L.R.,Vass, S.O.,Evans, A.,Searle, P.F.,White, S.A.,Hyde, E.I. Structure and Dynamics of Three Escherichia coli NfsB Nitro-Reductase Mutants Selected for Enhanced Activity with the Cancer Prodrug CB1954. Int J Mol Sci, 24:-, 2023 Cited by PubMed Abstract: 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. PubMed: 36983061DOI: 10.3390/ijms24065987 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.65 Å) |
Structure validation
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