title: Involvement of the cytochrome P450 system EthBAD in the N-deethoxymethylation of acetochlor by Rhodococcus sp. strain T3-1.
abstract: Acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)-acetamide] is a widely applied herbicide with potential carcinogenic properties. N-Deethoxymethylation is the key step in acetochlor biodegradation. N-Deethoxymethylase is a multicomponent enzyme that catalyzes the conversion of acetochlor to 2'-methyl-6'-ethyl-2-chloroacetanilide (CMEPA). Fast detection of CMEPA by a two-enzyme (N-deethoxymethylase-amide hydrolase) system was established in this research. Based on the fast detection method, a three-component enzyme was purified from Rhodococcus sp. strain T3-1 using ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular masses of the components of the purified enzyme were estimated to be 45, 43, and 11 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the results of peptide mass fingerprint analysis, acetochlor N-deethoxymethylase was identified as a cytochrome P450 system, composed of a cytochrome P450 oxygenase (43-kDa component; EthB), a ferredoxin (45 kDa; EthA), and a reductase (11 kDa; EthD), that is involved in the degradation of methyl tert-butyl ether. The gene cluster ethABCD was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). Resting cells of a recombinant E. coli strain showed deethoxymethylation activity against acetochlor. Subcloning of ethABCD showed that ethABD expressed in E. coli BL21(DE3) has the activity of acetochlor N-deethoxymethylase and is capable of converting acetochlor to CMEPA.
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Nanjing Agriculture University, Nanjing, People's Republic of China College of Bioscience and Bioengineering, Jiangxi Agriculture University, Nanchang, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Nanjing Agriculture University, Nanjing, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Nanjing Agriculture University, Nanjing, People's Republic of China.
authors.forename: Zhoukun
authors.initials: Z
authors.lastname: Li
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Nanjing Agriculture University, Nanjing, People's Republic of China.
authors.forename: Weiliang
authors.initials: W
authors.lastname: Dong
authors.affiliation: College of Food and Bioengineering, Henan University of Science and Technology, Luoyang, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Nanjing Agriculture University, Nanjing, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Nanjing Agriculture University, Nanjing, People's Republic of China czl@njau.edu.cn.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cui
mesh_terms: D001711:Biotransformation; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D016000:Cluster Analysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D006540:Herbicides / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010802:Phylogeny; D012240:Rhodococcus / Q000201:enzymology* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D014052:Toluidines / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D005288:Ferredoxins; D006540:Herbicides; D014052:Toluidines; C043377:acetochlor; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases