Characterization of Arylalkylamine N-Acyltransferase from Tribolium castaneum: An investigation into a potential next-generation insecticide target.
Characterization of Arylalkylamine N-Acyltransferase from Tribolium castaneum: An investigation into a potential next-generation insecticide target.
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DOI：
10.1021/acschembio.9b00973
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发表时间：
2020-01
影响因子：
4
通讯作者：
Brian G. O’Flynn;E. M. Lewandowski;Karin Claire Prins;Gabriela Suarez;Angelica N McCaskey;Nasha M Rios-Guzman;Ryan L. Anderson;Britney A. Shepherd;Ioannis Gelis;J. Leahy;Yu Chen;D. Merkler
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文献类型：
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作者：
Brian G. O’Flynn;E. M. Lewandowski;Karin Claire Prins;Gabriela Suarez;Angelica N McCaskey;Nasha M Rios-Guzman;Ryan L. Anderson;Britney A. Shepherd;Ioannis Gelis;J. Leahy;Yu Chen;D. Merkler
日益严重的杀虫剂抗药性问题意味着识别新型杀虫剂靶标变得前所未有的重要。芳基烷基胺 N-酰基转移酶 (AANAT) 被认为是一个潜在的新靶点。这些混杂的酶参与生物胺的 N-酰化形成 N-酰胺。在昆虫中，这个过程是黑化、角质层硬化、生物胺去除以及脂肪酸酰胺生物合成的关键步骤。每种 AANAT 同工型的独特性质表明，每种生物体都容纳了该生物体相对独有的离散 AANAT 组装体。这意味着杀虫剂设计具有很高的选择性潜力，同时也保持多药理学。这里介绍的是对世界上所有植物商品中最常见的次生害虫之一赤拟谷盗中发现的 AANAT 进行的彻底的动力学和结构分析。该酶名为 TcAANAT0，催化短链 N-酰基烷基胺的形成，其中短链酰基辅酶 A (C2-C10)、苯甲酰辅酶 A 和琥珀酰辅酶 A 充当酰基供体的角色。重组 TcAANAT0 从大肠杆菌中表达并纯化，用于研究催化动力学和化学机制。动力学机制是顺序机制，酰基辅酶A首先结合。 pH 速率曲线和定点诱变研究确定了对催化至关重要的氨基酸，提供了有关 TcAANAT0 化学机制的见解。获得了与乙酰辅酶 A 结合的 TcAANAT0 的晶体结构，揭示了有关其活性位点的有价值的信息。动力学分析和晶体学以及诱变和序列分析的结合揭示了一些可能针对 TcAANAT0 和其他 AANAT 的新型杀虫剂设计方法。
The growing issue of insecticide resistance has meant the identification of novel insecticide targets has never been more important. Arylalkylamine N-acyltransferases (AANATs) have been suggested as a potential new target. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. In insects, this process is a key step in melanism, hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each AANAT isoform characterized indicates each organism accommodates an assembly of discrete AANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology. Presented here is a thorough kinetic and structural analysis of AANAT found in one of the most common secondary pests of all plant commodities in the world, Tribolium castaneum. The enzyme, named TcAANAT0, catalyzes the formation of short-chain N-acylarylalkylamines, with short-chain acyl-CoAs (C2-C10), benzoyl-CoA, and succinyl-CoA functioning as the role of acyl-donor. Recombinant TcAANAT0 was expressed and purified from E. coli and was used to investigate the kinetic and chemical mechanism of catalysis. The kinetic mechanism is ordered sequential mechanism with the acyl-CoA binding first. pH-rate profiles and site-directed mutagenesis studies identified amino acids critical to catalysis, providing insights about the chemical mechanism of TcAANAT0. A crystal structure was obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. This combination of kinetic analysis, and crystallography, alongside mutagenesis, and sequence analysis shines light on some approaches possible to target TcAANAT0 and other AANATs for novel insecticide design.