The Properties of 5-Methyltetrahydrofolate Dehydrogenase (MetF1) and Its Role in the Tetrahydrofolate-Dependent Dicamba Demethylation System in Rhizorhabdus dicambivorans Ndbn-20

Abstract

The herbicide dicamba is initially degraded via the tetrahydrofolate (THF)-dependent demethylation system in Rhizorhabdus dicambivorans Ndbn-20. Two THF-dependent dicamba methyltransferase gene clusters, scaffold 50 and scaffold 66, were found in the genome of strain Ndbn-20. Each cluster contains a dicamba methyltransferase gene and three THF metabolism-related genes, namely, metF (coding for 5,10-CH₂-THF reductase), folD (coding for 5,10-CH₂-THF dehydrogenase-5,10-methenyl-THF cyclohydrolase), and purU (coding for 10-formyl-THF deformylase). In this study, reverse transcription-PCR (RT-PCR) results showed that only genes in scaffold 66, not those in scaffold 50, were transcribed in dicamba-cultured cells. The metF gene of scaffold 66 (metF1) was expressed in Escherichia coli BL21(DE3), and the product was purified as a His₆-tagged protein. Purified MetF1 was found to be a monomer and exhibited 5-CH₃-THF dehydrogenase activity in vitro The k_cat and K_m for 5-CH₃-THF were 0.23 s^-1 and 16.48 μM, respectively. However, 5,10-CH₂-THF reductase activity was not detected for MetF1 under the conditions tested. Gene disruption results showed that metF1 is essential for dicamba degradation, whereas folD1 is dispensable.IMPORTANCE There are several THF-dependent methyltransferase genes and THF-metabolic genes in the genome of R. dicambivorans Ndbn-20; however, which genes are involved in dicamba demethylation and the mechanism underlying THF regeneration remain unknown. This study revealed that scaffold 66 is responsible for dicamba demethylation and that MetF1 physiologically catalyzes the dehydrogenation of 5-CH₃-THF to 5,10-CH₂-THF in the THF-dependent dicamba demethylation system in R. dicambivorans Ndbn-20. Furthermore, the results showed that MetF1 differs from previously characterized MetF in phylogenesis, biochemical properties, and catalytic activity; e.g., MetF1 in vitro did not show 5,10-CH₂-THF reductase activity, which is the physiological function of Escherichia coli MetF. This study provides new insights into the mechanism of the THF-dependent methyltransferase system.

Keywords: 5-CH3-THF dehydrogenase activity; MetF1; THF regeneration pathway; THF-dependent dicamba demethylation system; enzymatic characteristics; gene disruption.

FIG 1

(A) Organization of the THF-dependent methyltransferase gene clusters. Shown are diagrams of scaffold 66, scaffold 50, scaffold 02, and other putative THF-metabolic genes in the Ndbn-20 genome. dmt and dmt50, dicamba methyltransferase genes; dmt02, methyltransferase gene; metF1 and metF, 5-CH₃-THF dehydrogenase and 5,10-CH₂-THF reductase genes; folD1 and folD, 5,10-CH₂-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase genes; purU1 and purU, formyl-THF deformylase genes. The locus tag for each gene in the Ndbn-20 genome (GenBank accession number CP023449) shown here consists of CMV14_ followed by the number in parentheses after the gene name. Arrows indicate the sizes and transcriptional directions of the genes. Lines below the gene cluster show the locations and sizes of the PCR fragments whose numbers correspond to lanes in panel B. (B) (I) Transcriptional analysis by RT-PCR of each methyltransferase gene and the THF-metabolic genes shown in panel A. (II) Agarose gel electrophoresis products obtained by RT-PCR primers using genomic DNA of strain Ndbn-20 as the template. (C) Proposed regeneration pathway of THF from 5-CH₃-THF during dicamba demethylation in R. dicambivorans Ndbn-20. The reactions catalyzed by FolD1 and PurU1 have not been confirmed by enzymatic studies.

FIG 2

Gel filtration of purified MetF1 and E. coli MetF. (A) Peak volumes of standard proteins: myosin (200.0 kDa; 10.00 ml) (a), β-galactosidase (116.0 kDa; 11.55 ml) (b), phosphorylase b (97.2 kDa; 12.38 ml) (c), bovine serum albumin (66.4 kDa; 13.46 ml) (d), and ovalbumin (44.3 kDa; 14.58 ml) (e). (B) Peak volume of native MetF1 (15.52 ml). (C) Peak volume of native E. coli MetF (11.84 ml). (D) Calibration line of standard proteins and determination of the molecular masses of MetF1 (33.4 kDa) and E. coli MetF (110.2 kDa).

FIG 3

HPLC analysis of the products generated during 5-CH₃-THF dehydrogenation by MetF1. (A) HPLC analysis of the product THF. (a1) THF standard; (a2) ascorbic acid standard; (a3) 5-CH₃-THF control without the addition of an enzyme; (a4) product generated during 5-CH₃-THF dehydrogenation by MetF1. The 5.46-min peak represents THF; the 3.11-min peak represents ascorbic acid added to the solvent or enzymatic mixture to maintain a reducing environment; and the 6.38-min peak represents 5-CH₃-THF. THF and 5-CH₃-THF were determined at 315 nm. (B) HPLC analysis of the product formaldehyde. (b1) 2,4-Dinitrophenylhydrazine standard; (b2) the derivative of formaldehyde and 2,4-dinitrophenylhydrazine; (b3) control in which 5-CH₃-THF but no enzyme was added to the reaction mixture, and no formaldehyde was produced; (b4) 5-CH₃-THF and MetF1 were added to the reaction mixture, and formaldehyde was produced. The 5.21-min (or 5.22-min) peak represents the derivative 2,4-dinitrobenzenehydrazone, and the 4.00-min peak represents 2,4-dinitrophenylhydrazine. 2,4-Dinitrophenylhydrazine and 2,4-dinitrobenzenehydrazone were determined at 365 nm.

FIG 4

Spectrophotometric changes during the determination of 5,10-CH₂-THF reductase activities for E. coli MetF (A) and MetF1 (B). The spectra were recorded every 30 s. The arrow indicates the direction of spectral changes.

FIG 5

Time course of dicamba degradation by the Ndbn-20ΔmetF1 mutant (A), wild-type Ndbn-20 (B), and the Ndbn-20ΔfolD1 mutant (C). Filled symbols, dicamba concentration; open symbols, OD₆₀₀. The data were derived from three independent measurements, and error bars indicate standard deviations.

FIG 6

Phylogenetic tree constructed on the basis of alignment of MetF1 with related MetF proteins. The multiple-alignment analysis was performed with ClustalX, v2.0, and the phylogenetic tree was constructed by the neighbor-joining method using MEGA 5.0. Bootstrap values (based on 1,000 replications) are indicated at branch nodes. Bar, 0.2 substitution per nucleotide position. Each item is arranged in the following order: protein name, protein accession number, and organism name. The four MetF proteins from R. dicambivorans Ndbn-20 are underlined. The locus tags of the genes encoding the MetF proteins with NCBI Protein database accession numbers WP_096616645.1 and WP_066959618.1 are CMV14_12645 (in scaffold 50) and CMV14_03770 (in scaffold 02), respectively, and that for MetF with accession number WP_066968257.1 is CMV14_23575.