Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China.
Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China
Appl Environ Microbiol. 2020 Dec 17;87(1). doi: 10.1128/AEM.02005-20.
Methomyl {bis[1-methylthioacetaldehyde--(-methylcarbamoyl)oximino]sulfide} is a highly toxic oxime carbamate insecticide. Several methomyl-degrading microorganisms have been reported so far, but the role of specific enzymes and genes in this process is still unexplored. In this study, a protein annotated as a carbamate C-N hydrolase was identified in the methomyl-degrading strain MDW-2, and the encoding gene was termed A comparative analysis between the mass fingerprints of AmeH and deduced proteins of the strain MDW-2 genome revealed AmeH to be a key enzyme of the detoxification step of methomyl degradation. The results also demonstrated that AmeH was a functional homodimer with a subunit molecular mass of approximately 34 kDa and shared the highest identity (27%) with the putative formamidase from ATCC 24843. AmeH displayed maximal enzymatic activity at 50°C and pH 8.5. and of AmeH for methomyl were 87.5 μM and 345.2 s, respectively, and catalytic efficiency (/ ) was 3.9 μM s Phylogenetic analysis revealed AmeH to be a member of the FmdA_AmdA superfamily. Additionally, five key amino acid residues (162, 164, 191, 193, and 207) of AmeH were identified by amino acid variations. Based on the structural characteristic, carbamate insecticides can be classified into oxime carbamates (methomyl, aldicarb, oxamyl, etc.) and -methyl carbamates (carbaryl, carbofuran, isoprocarb, etc.). So far, research on the degradation of carbamate pesticides has mainly focused on the detoxification step and hydrolysis of their carbamate bond. Several genes, such as , , , and , and their encoding enzymes have also been reported to be involved in the detoxification step. However, none of these enzymes can hydrolyze methomyl. In this study, a carbamate C-N hydrolase gene, , responsible for the detoxification step of methomyl in strain MDW-2 was cloned and the key amino acid sites of AmeH were investigated. These findings provide insight into the microbial degradation mechanism of methomyl.
涕灭威{双[1-甲硫基乙醛-(-甲基氨基甲酰)肟基]硫化物}是一种剧毒肟基氨基甲酸酯类杀虫剂。到目前为止，已经报道了几种涕灭威降解微生物，但该过程中特定酶和基因的作用仍未得到探索。在这项研究中，在涕灭威降解菌株 MDW-2 中鉴定出一种被注释为氨基甲酸酯 C-N 水解酶的蛋白质，并将其编码基因命名为 AmeH。对 AmeH 和菌株 MDW-2 基因组推导蛋白的质量指纹图谱进行比较分析，表明 AmeH 是涕灭威降解解毒步骤的关键酶。结果还表明，AmeH 是一种功能性同源二聚体，亚基分子量约为 34kDa，与 ATCC 24843 中的假定甲酰胺酶具有最高的同一性（27%）。AmeH 在 50°C 和 pH 8.5 时表现出最大的酶活性。AmeH 对涕灭威的 和 值分别为 87.5 μM 和 345.2 s，催化效率（/）为 3.9 μM s。系统发育分析表明，AmeH 是 FmdA_AmdA 超家族的成员。此外，通过氨基酸变异鉴定出 AmeH 的五个关键氨基酸残基（162、164、191、193 和 207）。根据结构特征，氨基甲酸酯类杀虫剂可分为肟基氨基甲酸酯类（涕灭威、涕灭砜、灭多威等）和-甲基氨基甲酸酯类（西维因、呋喃丹、异丙威等）。到目前为止，对氨基甲酸酯类农药的降解研究主要集中在其氨基甲酸酯键的解毒和水解步骤。已经报道了几个基因，如 、 、 、 和 ，以及它们编码的酶，参与了解毒步骤。然而，这些酶都不能水解涕灭威。在本研究中，克隆了负责 MDW-2 菌株中涕灭威解毒步骤的氨基甲酸酯 C-N 水解酶基因 ，并研究了 AmeH 的关键氨基酸位点。这些发现为涕灭威的微生物降解机制提供了深入的了解。
