Dataset Information

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??M, 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.

SUBMITTER: Yao S

PROVIDER: S-EPMC6689297 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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

Yao Shigang S Chen Le L Yang Zhou Z Yao Li L Zhu Jianchun J Qiu Jiguo J Wang Guoxiang G He Jian J

Journal of bacteriology 20190808 17

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 dehyd ...[more]

PMID: 31209079

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Project description:Sphingomonas sp. strain Ndbn-20 degrades and utilizes the herbicide dicamba as its sole carbon and energy source. In the present study, a tetrahydrofolate (THF)-dependent dicamba methyltransferase gene, dmt, was cloned from the strain, and three other genes, metF, dhc, and purU, which are involved in THF metabolism, were found to be located downstream of dmt A transcriptional study revealed that the four genes constituted one transcriptional unit that was constitutively transcribed. Lysates of cells grown with glucose or dicamba exhibited almost the same activities, which further suggested that the dmt gene is constitutively expressed in the strain. Dmt shared 46% and 45% identities with the methyltransferases DesA and LigM from Sphingomonas paucimobilis SYK-6, respectively. The purified Dmt catalyzed the transfer of methyl from dicamba to THF to form the herbicidally inactive metabolite 3,6-dichlorosalicylic acid (DCSA) and 5-methyl-THF. The activity of Dmt was inhibited by 5-methyl-THF but not by DCSA. The introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba. In conclusion, this study identified a THF-dependent dicamba methyltransferase, Dmt, with potential applications for the genetic engineering of dicamba-resistant crops.Dicamba is a very important herbicide that is widely used to control more than 200 types of broadleaf weeds and is a suitable target herbicide for the engineering of herbicide-resistant transgenic crops. A study of the mechanism of dicamba metabolism by soil microorganisms will benefit studies of its dissipation, transformation, and migration in the environment. This study identified a THF-dependent methyltransferase, Dmt, capable of catalyzing dicamba demethylation in Sphingomonas sp. Ndbn-20, and a preliminary study of its enzymatic characteristics was performed. Introduction of a codon-optimized dmt gene into Arabidopsis thaliana enhanced resistance against dicamba, suggesting that the dmt gene has potential applications for the genetic engineering of herbicide-resistant crops.

Project description:The herbicide dicamba is initially demethylated to 3,6-dichlorosalicylate (3,6-DCSA) in Rhizorhabdus dicambivorans Ndbn-20 and is subsequently 5-hydroxylated to 3,6-dichlorogentisate (3,6-DCGA). In the present study, two glutathione-dependent 3,6-DCGA dehalogenases, DsmH1 and DsmH2, were identified in strain Ndbn-20. DsmH2 shared a low identity (only 31%) with the tetrachlorohydroquinone (TCHQ) dehalogenase PcpC from Sphingobium chlorophenolicum ATCC 39723, while DsmH1 shared a high identity (79%) with PcpC. In the phylogenetic tree of related glutathione S-transferases (GSTs), DsmH1 and DsmH2, together with PcpC and the 2,5-dichlorohydroquinone dehalogenase LinD, formed a separate clade. DsmH1 and DsmH2 were synthesized in Escherichia coli BL21 and purified as His-tagged enzymes. Both enzymes required glutathione (GSH) as a cofactor and could 6-dechlorinate 3,6-DCGA to 3-chlorogentisate in vitro DsmH2 had a significantly higher catalytic efficiency toward 3,6-DCGA than DsmH1. Transcription and disruption analysis revealed that DsmH2 but not DsmH1 was responsible for the 6-dechlorination of 3,6-DCGA in strain Ndbn-20 in vivo Furthermore, we propose a novel eta class of GSTs to accommodate the four bacterial dehalogenases PcpC, LinD, DsmH1, and DsmH2.IMPORTANCE Dicamba is an important herbicide, and its use and leakage into the environment have dramatically increased since the large-scale planting of genetically modified (GM) dicamba-resistant crops in 2015. However, the complete catabolic pathway of dicamba has remained unknown, which limits ecotoxicological studies of this herbicide. Our previous study revealed that 3,6-DCGA was an intermediate of dicamba degradation in strain Ndbn-20. In this study, we identified two glutathione-dependent 3,6-DCGA dehalogenases, DsmH1 and DsmH2, and demonstrated that DsmH2 is physiologically responsible for the 6-dechlorination of 3,6-DCGA in strain Ndbn-20. GSTs play an important role in the detoxification and degradation of a variety of endogenous and exogenous toxic compounds. On the basis of their sequence identities, phylogenetic status, and functions, the four bacterial GSH-dependent dehalogenases (PcpC, LinD, DsmH1, and DsmH2) were reclassified as a new eta class of GSTs. This study helps us to elucidate the microbial catabolism of dicamba and enhances our understanding of the diversity and functions of GSTs.

Dataset Information

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

Publications

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

Similar Datasets

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