The main metabolic product of the pyridinecarboxamide insecticide flonicamid, -(4-trifluoromethylnicotinyl)glycinamide (TFNG-AM), has been shown to have very high mobility in soil, leading to its accumulation in the environment. Catabolic pathways of flonicamid have been widely reported, but few studies have focused on the metabolism of TFNG-AM. Here, the rapid transformation of TFNG-AM and production of the corresponding acid product -(4-trifluoromethylnicotinoyl) glycine (TFNG) by the plant growth-promoting bacterium CGMCC 4969 were investigated. With TFNG-AM at an initial concentration of 0.86 mmol/L, 90.70 % was transformed by CGMCC 4969 resting cells within 20 d, with a degradation half-life of 4.82 d. A novel amidase that potentially mediated this transformation process, called AmiD, was identified by bioinformatic analyses. The gene encoding was cloned and expressed recombinantly in , and the enzyme AmiD was characterized. Key amino acid residue Val154, which is associated with the catalytic activity and substrate specificity of signature family amidases, was identified for the first time by homology modeling, structural alignment, and site-directed mutagenesis analyses. When compared to wild-type recombinant AmiD, the mutant AmiD V154G demonstrated a 3.08-fold increase in activity toward TFNG-AM. The activity of AmiD V154G was greatly increased toward aromatic -phenylalanine amides, heterocyclic TFNG-AM and IAM, and aliphatic asparagine, whereas it was dramatically lowered toward benzamide, phenylacetamide, nicotinamide, acetamide, acrylamide, and hexanamid. Quantitative PCR analysis revealed that AmiD may be a substrate-inducible enzyme in CGMCC 4969. The mechanism of transcriptional regulation of AmiD by a member of the AraC family of regulators encoded upstream of the gene was preliminarily investigated. This study deepens our understanding of the mechanisms of metabolism of toxic amides in the environment, providing new ideas for microbial bioremediation.

中文翻译：

---

Asp-tRNAAsn/Glu-tRNAGln 酰胺转移酶 A 亚基样酰胺酶介导 Variovorax boronicumulans CGMCC 4969 降解杀虫剂氟啶酰胺

吡啶甲酰胺杀虫剂氟啶虫酰胺的主要代谢产物-(4-三氟甲基烟基)甘氨酰胺(TFNG-AM)已被证明在土壤中具有非常高的迁移性，导致其在环境中积累。氟啶酰胺的分解代谢途径已被广泛报道，但很少有研究关注 TFNG-AM 的代谢。在此，研究了植物促生长细菌CGMCC 4969对TFNG-AM的快速转化以及相应的酸产物-(4-三氟甲基烟酰)甘氨酸(TFNG)的生产。 TFNG-AM初始浓度为0.86 mmol/L时，20 d内CGMCC 4969静息细胞转化率为90.70 %，降解半衰期为4.82 d。通过生物信息学分析鉴定出一种可能介导这一转化过程的新型酰胺酶，称为 AmiD。在 中克隆并重组表达了编码基因，并对 AmiD 酶进行了表征。关键氨基酸残基 Val154 与特征家族酰胺酶的催化活性和底物特异性相关，首次通过同源建模、结构比对和定点诱变分析进行鉴定。与野生型重组 AmiD 相比，突变体 AmiD V154G 的 TFNG-AM 活性增加了 3.08 倍。 AmiD V154G 对芳香族苯丙氨酸酰胺、杂环 TFNG-AM 和 IAM 以及脂肪族天冬酰胺的活性大大增加，而对苯甲酰胺、苯乙酰胺、烟酰胺、乙酰胺、丙烯酰胺和己酰胺的活性显着降低。定量PCR分析表明AmiD可能是CGMCC 4969中的底物诱导酶。初步研究了该基因上游编码的AraC家族成员对AmiD转录调控的机制。该研究加深了我们对环境中有毒酰胺代谢机制的认识，为微生物生物修复提供了新思路。