Acetamiprid, a chloronicotinyl neonicotinoid insecticide, is among the most commonly used insecticides worldwide, and its environmental fate has caused considerable concern. The compound 1-(6-chloropyridin-3-yl)-N-methylmethanamine (IM 1-4) has been reported as the main intermediate during acetamiprid catabolism in microorganisms, honeybees and spinach. However, the molecular mechanism underlying the hydrolysis of acetamiprid to IM 1-4 has not yet been elucidated. In this study, a novel amidase (AceAB) that initially hydrolyzes the C-N bond of acetamiprid to generate IM 1-4 was purified and characterized from the acetamiprid-degrading strain Pigmentiphaga sp. D-2. Based on peptide profiling of the purified AceAB and the draft genome sequence of strain D-2, aceA (372 bp) and aceB (2,295 bp) encoding the α and β subunits of AceAB, respectively, were cloned and found to be necessary for acetamiprid hydrolysis in strain D-2. The characteristics of AceAB were also systematically investigated. Though AceA and AceB showed 35%-56% identities to the α and β subunits of the N,N-dimethylformamidase from Paracoccus aminophilus, AceAB was specific for the hydrolysis of acetamiprid and showed no activities to N,N-dimethylformamide or its structural analogs.

IMPORTANCE Acetamiprid, belonging to the top world-widely used neonicotinoid insecticides, is one of the most important commercial insecticides. Due to its extensive use, the environmental fate of acetamiprid, especially the microbial degradation of acetamiprid, has caused considerable concern. Although the catabolic pathways of acetamiprid in microorganisms have been extensively studied, the molecular mechanisms underlying acetamiprid biodegradation (except for a nitrile hydratase) remain largely unknown, and the enzyme responsible for the biotransformation of acetamiprid to its main intermediate IM 1-4 has not yet been elucidated. The amidase AceAB and its encoding genes aceA and aceB, characterized in this study, was found to be necessary and specific for the initial hydrolysis of C-N bond of acetamiprid to generate IM 1-4 in Pigmentiphaga sp. strain D-2. The finding of the novel amidase AceAB will greatly enhance our understanding of the microbial catabolism of the widely used insecticide acetamiprid on the molecular level.

乙草胺，一种氯烟碱新烟碱类杀虫剂，是全世界最常用的杀虫剂之一，其环境命运引起了极大的关注。据报道，化合物1-（6-氯吡啶-3-基）-N-甲基甲胺（IM 1-4）是在微生物，蜜蜂和菠菜的乙酰胺分解代谢过程中的主要中间体。然而，尚未阐明将乙酰胺水解为IM 1-4的分子机制。在这项研究中，一种新型的酰胺酶（AceAB）首先水解了乙酰胺的CN键以生成IM 1-4，并从降解乙酰胺的菌株Pigmentiphaga sp。中进行了鉴定。D-2。基于该纯化AceAB的肽分析和应变d-2的基因组序列草图ACEA分别克隆了AceAB的α和β亚基（372 bp）和aceB（2295 bp），发现它们对菌株D-2中的乙酰胺水解是必需的。还对AceAB的特性进行了系统研究。尽管AceA和AceB与嗜热副球菌N，N-二甲基甲酰胺酶的α和β亚基显示35％-56％的同一性，但AceAB对乙酰胺的水解具有特异性，对N，N-二甲基甲酰胺或其结构类似物没有活性。

重要信息乙草胺属于世界上使用最广泛的新烟碱类杀虫剂，是最重要的商业杀虫剂之一。由于其广泛使用，对乙酰胺的环境命运，特别是对乙酰胺的微生物降解，引起了极大的关注。尽管已广泛研究了扑热息痛在微生物中的分解代谢途径，但基本上不知道引起扑热息痛生物降解的分子机制（除腈水合酶外），并且负责将扑热息痛生物转化为其主要中间体IM 1-4的酶尚未确定。被阐明。酰胺酶AceAB及其编码基因aceA和aceB，本研究中发现的，被发现对于扑热息痛的CN键初始水解以在Pigmentiphaga sp。中生成IM 1-4是必要的和特定的。菌株D-2。新型酰胺酶AceAB的发现将在分子水平上极大地增进我们对广泛使用的杀虫剂对乙酰氨基酚的微生物分解代谢的理解。