A Bifunctional Cytochrome P450 Enzyme Involved in the O-Dealkylation or N-Dealkoxymethylation of Chloroacetanilide Herbicides in Rhodococcus sp. B2

Abstract

Background: The chloroacetamide herbicides pretilachlor is an emerging pollutant, Due to the large amount of use, its presence in the environment threatens to human health. However, the molecular mechanism of pretilachlor degradation is unknown. Results: Now, Rhodococcus sp. B2 was isolated from rice field and found to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, 50 mg/L initial substrate, pH 6.98, and 30.1°C. One novel metabolite was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, comprising a gene cluster (EthRABCDB2), was absent in the mutant strain TB2 which could not degrade pretilachlor. The Eth gene system, encodes an AraC/XylS family transcriptional regulator, a ferredoxin reductase, a cytochrome P-450 monooxygenase, a ferredoxin and a 10-kDa unknown protein. Complementation of EthABCDB2 and EthABDB2, but not EthABCB2 in strain TB2 restored its activity against chloroacetamide herbicides. The codon of EthABCDB2 was optimized, expressed in Escherichia coli, and purified utilizing Ni-affinity chromatography. A mixture of EthABCDB2 or EthABDB2 but not EthABCB2 catalyzed the N-dealkoxymethylation activity toward alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation activity toward pretilachlor, revealing that EthD acted as a ferredoxin in strain B2. EthABDB2 displayed maximal activity at 30 °C and pH 7.5. Conclusions: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation or N-dealkoxymethylation of pretilachlor and propisochlor. And the results provide microbial resource for the remediation of chloroacetamide herbicides-contaminated sites,