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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