Escherichia coli
nitroreductase (NTR) is a
flavoprotein that reduces a variety of
quinone and nitroaromatic substrates. Among these substrates is the
prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (
CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with
CB1954 has entered clinical trials for virus-directed
enzyme-
prodrug therapy of
cancer. Enhancing the catalytic efficiency of NTR for
CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to
CB1954 in an E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for
CB1954 and for the
antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for
CB1954, and, apart from N71S, the enhancement is selective for
CB1954 over
nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of
CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue
nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the
FMN cofactor, while mutations at T41 and F124 affect the interactions with
nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with
CB1954 over
nitrofurazone than either of these, and the highest specificity constant for
CB1954 of all the mutations studied.