Involvement of the Cytochrome P450 System EthBAD in the N -Deethoxymethylation of Acetochlor by Rhodococcus sp. Strain T3-1

Abstract

c 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. Chloroacetanilide herbicides are a class of important herbicides used worldwide. The chloroacetanilide herbicide acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)-acetamide] is a selective preemergent herbicide that has been used to effectively control broadleaf weeds and annual grasses in corn fields for almost 40 years (1). The excessive and frequent application of acetochlor may result in high levels of acetochlor residues, which have been detected in ground and surface waters and negatively impact both the environment and agricultural ecosystems (2). The U.S. Environmental Protection Agency (EPA) has classified acetochlor as a B-2 carcinogen and a probable human carcinogen (3). Studies of different soil types treated with acetochlor have demonstrated that 2=-methyl-6=-ethyl-2-chloroacetanilide (CMEPA), a compound derived from acetochlor N-deethoxymethylation, is a major product of the microbial degradation process (1, 3-5).We previously isolated an efficient acetochlor N-deethoxymethylation strain, T3-1, from a microbial consortium that could mineralize acetochlor completely. The strain was identified as a Rhodococcus sp. (1). The biochemical pathway of acetochlor degradation by the three bacteria in the consortium was proposed to be as follows: acetochlor to CMEPA by Rhodococcus sp. strain T3-1, CMEPA to 2-methyl-6-ethyl aniline (MEA) by Delftia sp. strain T3-6, and MEA by Sphingobium sp. strain MEA3-1, based on the identified degradation intermediates (1). In this study, a three-...

A Tetrahydrofolate-Dependent Methyltransferase Catalyzing the Demethylation of Dicamba in Sphingomonas sp. Strain Ndbn-20

“…The two genes were induced, and the C-terminal His 6 -tagged proteins were purified using the method described by Wang et al (29). The molecular weights were determined by SDS-PAGE and Coomassie blue staining, and the protein concentrations were quantified by the Bradford method using bovine serum albumin as the standard (30,31).…”

The properties of 5-methyltetrahydrofolate dehydrogenase (MthfD) and its role in the tetrahydrofolate (THF)-dependent dicamba demethylation system inRhizorhabdus dicambivoransNdbn-20

“…Demethylation is usually the initial and critical step in the microbial degradation of methyl-substituted compounds. To date, three types of demethylases from aerobic bacteria have been reported: Rieske non-heme iron oxygenase (RHO) (11, 12), cytochrome P450 (13, 14) and tetrahydrofolate (THF)-dependent methyltransferase (6, 15–21). Both RHO and P450 are multi-component monooxygenase systems with NAD(P)H as cofactor, these demethylases add a molecule of oxygen to the methyl group, resulting in removal of the methyl group from the substrate to form formaldehyde.…”

Metabolic Pathway Involved in 2-Methyl-6-Ethylaniline Degradation by Sphingobium sp. Strain MEA3-1 and Cloning of the Novel Flavin-Dependent Monooxygenase System meaBA

“…Genomic DNA was extracted and purified from strain MEA3-1 via high-salt precipitation (11). Three PCR fragments (1,224 bp, 2,069 bp, and 6,247 bp) containing the 60-bp potential promoter region of meaB were amplified using the primer pairs MeaAp-F/MeaA-R, MeaBp-F/ MeaA-R, and MeaBp-F/Orf4-R (Table 2), designated meaA, meaBA, and meaBA-orf1-4 (i.e., meaBA with orf1 to orf4), respectively.…”

“…T3-1 and Delftia sp. T3-6 (11,12). In this study, we clarified the partial metabolic pathway responsible for MEA degradation and cloned a novel flavin-dependent monooxygenase system, MeaBA, involved in MEA degradation.…”

“…A cytochrome P450 system, EthBAD, was reported to be involved in the N-deethoxymethylation of acetochlor by Rhodococcus sp. strain T3-1 (11). Two genes, cmeH and damH, were cloned from Sphingobium quisquiliarum strain DC-2 and Delftia sp.…”