Altering the Regioselectivity of a Nitroreductase in the Synthesis of Arylhydroxylamines by Structure‐Based Engineering

Nitroreductases have great potential for the highly efficient reduction of aryl nitro compounds to arylhydroxylamines. However, regioselective reduction of the desired nitro group in polynitroarenes is still a challenge. Here, we describe the structure‐based engineering of Escherichia coli nitroreductase NfsB to alter its regioselectivity, in order to achieve reduction of a target nitro group. When 2,4‐dinitrotoluene was used as the substrate, the wild‐type enzyme regioselectively reduced the 4‐NO₂ group, but the T41L/N71S/F124W mutant primarily reduced the 2‐NO₂ group, without loss of activity. The crystal structure of T41L/N71S/F124W and docking experiments indicated that the regioselectivity change (from 4‐NO₂ to 2‐NO₂) might result from the increased hydrophobicity of residues 41 and 124 (proximal to FMN) and conformational changes in residues 70 and 124.