The Properties of 5-Methyltetrahydrofolate Dehydrogenase (MetF1) and Its Role in the Tetrahydrofolate-Dependent Dicamba Demethylation System in Rhizorhabdus dicambivorans Ndbn-20
The Properties of 5-Methyltetrahydrofolate Dehydrogenase (MetF1) and Its Role in the Tetrahydrofolate-Dependent Dicamba Demethylation System in Rhizorhabdus dicambivorans Ndbn-20
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5-甲基四氢叶酸脱氢酶 (MetF1) 的特性及其在二氢叶酸依赖性麦草畏去甲基化系统中的作用 Rhizorhabdus dicambivorans Ndbn-20
DOI：
10.1128/jb.00096-19
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发表时间：
2019
影响因子：
3.2
通讯作者：
He Jian
中科院分区：
文献类型：
--
作者：
Yao Shigang;Chen Le;Yang Zhou;Yao Li;Zhu Jianchun;Qiu Jiguo;Wang Guoxiang;He Jian
在双目根霉NDBN-20中，除草剂麦草畏最初是通过依赖四氢叶酸(THF)的去甲基化系统降解的。在菌株NDBN-20基因组中发现了两个依赖于四氢呋喃的Dicamba甲基转移酶基因簇，即scaffold 50和scaffold 66。每个簇包含一个Dicamba甲基转移酶基因和三个与THF代谢相关的基因，即metF(编码5，10-CH2-THF还原酶)、Fold(编码5，10-CH2-THF脱氢酶-5，10-亚甲基-THF环水解酶)和PurU(编码10-甲酰-THF脱甲酰基酶)。在本研究中，逆转录-聚合酶链式反应(RT-PCR)结果显示，只有支架66中的基因，而不是支架50中的那些基因，在马唐培养的细胞中转录。在大肠杆菌BL21(DE3)中表达了支架66(MetF1)的etF基因，并将其纯化为His6标记的蛋白。纯化的MetF1是一种单体，体外具有5-CH3-THF脱氢酶活性。5-CH_3-THF的Kcat和Km分别为0.23 S−1和16.48 μM。然而，在所测试的条件下，没有检测到MetF1的5，10-CH2-THF还原酶活性。基因阻断结果表明，MetF1对麦草畏的降解是必需的，而FoleD1是必需的。重要的是，DDBN-20的基因组中有几个依赖于四氢呋喃的甲基转移酶基因和四氢呋喃代谢基因，但哪些基因参与了麦草畏去甲基化以及四氢呋喃的再生机制尚不清楚。这项研究揭示了支架66负责Dicamba去甲基化，并且MetF1在依赖THF的Dicamba去甲基化系统中生理地催化5-CH3-THF脱氢为5，10-CH2-THF。此外，结果表明，MetF1在系统发育、生化性质和催化活性等方面与以前鉴定的MetF有所不同，例如MetF1在体外不显示5，10-CH2-THF还原酶活性，而这是大肠杆菌MetF的生理功能。这项研究为THF依赖的甲基转移酶系统的机制提供了新的见解。
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), andpurU(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. ThemetFgene 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 activityin vitro. ThekcatandKmfor 5-CH3-THF were 0.23 s−1and 16.48 μM, respectively. However, 5,10-CH2-THF reductase activity was not detected for MetF1 under the conditions tested. Gene disruption results showed thatmetF1is essential for dicamba degradation, whereasfolD1is dispensable.IMPORTANCEThere 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., MetF1in vitrodid 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.