A novel pathway for initial biotransformation of dinitroaniline herbicide butralin from a newly isolated bacterium Sphingopyxis sp. strain HMH

摘要

Butralin (N -secButyl-4-tert-butyl-2,6-dinitroaniline) is a highly persistent dinitroaniline herbicide frequently detected in the environment. In this study, butralin-degrading soil bacterium, Sphingopyxis sp. strain HMH was isolated from agricultural soil samples. Based on whole genome sequence analysis of the strain HMH, the gene encoding a nitroreductase NfnB was identified and expressed in Escherichia coli (E. coli), and protein was purified to homogeneity. NfnB is a flavin-nitroreductase, found to be a functional tetramer, composed of subunit mo-lecular mass of 25 kDa. The metabolites from butralin degradation by strain HMH and purified NfnB were identified using ultra performance liquid chromatography high resolution mass spectrometry (UPLC-HRMS), and a novel mechanism of butralin degradation was proposed. NfnB selectively nitro-reduced butralin into N (sec Butyl)-4-(tert-butyl)-6-nitrobenzene1,2-diamine, followed by formation of 5-(tert-Butyl)-3 -nitrobenzene-1,2-diamine and butanone by Ndealkylation through possible hydroxylation reaction onto the carbon linked amine of the N-(sec-Butyl) moiety. In our study, we could not detect the hydroxylated product 2-(2-Amino-4-tert-butyl 6-nitrophenylamino)-butan-2-ol) (carbinolamine), instead its Schiff base product (E)-2-(Butan-2-yildenea mino)-5(tert-butyl)-3-nitroaniline was detected. The release of butanone was further confirmed by derivatization with 2,4dinitrophenylhydrazine (DNPH) followed by MS analysis. In conclusion, this study explores a novel multi-functional flavinnitroreductase family enzyme NfnB, catalyzing unique and sequential nitror-eduction and N-dealkylation through oxidative hydroxylation of dinitroaniline herbicide butralin.