8og3 | pdb_00008og3

E. coli NfsB triple mutant T41L/N71S/F124T bound to citrate

spinqualitylabelsall models 8og3, resolution 2.09Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 8og3 is a 2 chain structure with sequence from Escherichia coli. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: CIT, EDO, FMN Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function NFSB_ECOLI Reduction of a variety of nitroaromatic compounds using NADH (and to lesser extent NADPH) as source of reducing equivalents; two electrons are transferred. Capable of reducing nitrofurazone, quinones and the anti-tumor agent CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The reduction of CB1954 results in the generation of cytotoxic species. Publication Abstract from PubMed 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. Structure and Dynamics of Three Escherichia coli NfsB Nitro-Reductase Mutants Selected for Enhanced Activity with the Cancer Prodrug CB1954.,Day MA, Christofferson AJ, Anderson JLR, Vass SO, Evans A, Searle PF, White SA, Hyde EI Int J Mol Sci. 2023 Mar 22;24(6):5987. doi: 10.3390/ijms24065987. PMID:36983061 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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