5-甲基四氢叶酸脱氢酶（MetF1）的性质及其在 Rhizorhabdus dicambivorans Ndbn-20 中四氢叶酸依赖的麦草畏去甲基化系统中的作用。
The Properties of 5-Methyltetrahydrofolate Dehydrogenase (MetF1) and Its Role in the Tetrahydrofolate-Dependent Dicamba Demethylation System in Rhizorhabdus dicambivorans Ndbn-20.
机构信息
Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu, China.
School of Marine and Biological Engineering, Yancheng Teachers University, Yancheng, Jiangsu, China.
出版信息
J Bacteriol. 2019 Aug 8;201(17). doi: 10.1128/JB.00096-19. Print 2019 Sep 1.
The herbicide dicamba is initially degraded via the tetrahydrofolate (THF)-dependent demethylation system in 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, (coding for 5,10-CH-THF reductase), (coding for 5,10-CH-THF dehydrogenase-5,10-methenyl-THF cyclohydrolase), and (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 gene of scaffold 66 () was expressed in BL21(DE3), and the product was purified as a His-tagged protein. Purified MetF1 was found to be a monomer and exhibited 5-CH-THF dehydrogenase activity The and for 5-CH-THF were 0.23 s and 16.48 μM, respectively. However, 5,10-CH-THF reductase activity was not detected for MetF1 under the conditions tested. Gene disruption results showed that is essential for dicamba degradation, whereas is dispensable. There are several THF-dependent methyltransferase genes and THF-metabolic genes in the genome of 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-CH-THF to 5,10-CH-THF in the THF-dependent dicamba demethylation system in Ndbn-20. Furthermore, the results showed that MetF1 differs from previously characterized MetF in phylogenesis, biochemical properties, and catalytic activity; e.g., MetF1 did not show 5,10-CH-THF reductase activity, which is the physiological function of MetF. This study provides new insights into the mechanism of the THF-dependent methyltransferase system.
该除草剂二甲苯草胺最初通过 Ndbn-20 中的四氢叶酸 (THF) 依赖性去甲基化系统降解。在 Ndbn-20 的基因组中发现了两个 THF 依赖性二甲苯草胺甲基转移酶基因簇，支架 50 和支架 66。每个簇都包含一个二甲苯草胺甲基转移酶基因和三个 THF 代谢相关基因，即 （编码 5,10-CH-THF 还原酶）、 （编码 5,10-CH-THF 脱氢酶-5,10-亚甲基-THF 环水解酶）和 （编码 10-甲酰基-THF 变形酶）。在这项研究中，逆转录 PCR (RT-PCR) 结果表明，只有支架 66 中的基因，而不是支架 50 中的基因，在二甲苯草胺培养的细胞中转录。支架 66 的 基因（）在 BL21(DE3) 中表达，并作为 His 标记蛋白进行纯化。纯化的 MetF1 被发现为单体，并表现出 5-CH-THF 脱氢酶活性。MetF1 对 5-CH-THF 的 和 分别为 0.23 s 和 16.48 μM。然而，在测试的条件下，MetF1 没有检测到 5,10-CH-THF 还原酶活性。基因敲除结果表明，对于二甲苯草胺降解，是必不可少的，而 是可有可无的。在 Ndbn-20 的基因组中有几个 THF 依赖性甲基转移酶基因和 THF 代谢基因；然而，哪些基因参与二甲苯草胺去甲基化以及 THF 再生的机制仍不清楚。本研究表明，支架 66 负责二甲苯草胺去甲基化，MetF1 生理上催化 5-CH-THF 在 Ndbn-20 中 THF 依赖性二甲苯草胺去甲基化系统中的脱氢反应生成 5,10-CH-THF。此外，结果表明，MetF1 在系统发育、生化特性和催化活性方面与先前表征的 MetF 不同；例如，MetF1 没有表现出 5,10-CH-THF 还原酶活性，这是 MetF 的生理功能。本研究为 THF 依赖性甲基转移酶系统的机制提供了新的见解。
Zotero 插件