Structural basis of Escherichia coli nitroreductase NfsB triple mutants engineered for improved activity and regioselectivity toward the prodrug CB1954

•The single mutant F124W regioselectively reduces the 4-NO2 group of CB1954.•Mutations F123A and T41L increase the kcat values in two different ways.•Two triple mutants were obtained with superior activity and regioselectivity.

Escherichia coli nitroreductase NfsB coupled with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] has great anti-cancer potential. However, its efficacy is limited by the low catalytic efficiency of NfsB against CB1954. In this paper, we show that substitution with a tryptophan at residue 124 strongly increases the selectivity toward the 4-NO2 group of CB1954, generating the more cytotoxic 4-hydroxylamine product. To further improve the activity, the F124W mutation and three previously reported beneficial mutations were combined randomly into double and triple mutants. Steady-state kinetic studies showed that they all exhibited enhanced activity toward CB1954. Two triple mutants, T41L/N71S/F124W and F123A/N71S/F124W, show a 9.2–17.2-fold increase in kcat/Km compared with the wild-type and selectively reduce the 4-NO2 group of CB1954. The crystal structure of F123A/N71S/F124W was resolved. Comparison with the reported NfsB structures revealed that the F124W mutation may provide a stronger hydrophobic interaction with the aziridinyl group of CB1954, which was in favor of the reduction of the 4-NO2 group; while the F123A and T41L mutations increased the kcat values in two different ways, leading to improved enzyme activity.

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