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.
School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian-116023 (China).
Jing
J
Bai
School of Software Technology, Dalian University of Technology, 321 Tuqiang Street, Development Zone, Dalian (China).
Yong
Y
Zhou
School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian-116023 (China).
Qi
Q
Chen
School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian-116023 (China).
Qing
Q
Yang
School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian-116023 (China). junyang@dlut.edu.cn.
Jun
J
Yang
D020134:Catalytic Domain; D004136:Dinitrobenzenes / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019811:Hydroxylamine / Q000138:chemical synthesis* / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009601:Nitroreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
D004136:Dinitrobenzenes; D029968:Escherichia coli Proteins; D019811:Hydroxylamine; C016403:2,4-dinitrotoluene; C116054:NfsB protein, E coli; D009601:Nitroreductases
arylhydroxylamines; enzyme catalysis; nitroreductases; protein engineering; regioselectivity