The COOH terminus of arylamine N-acetyltransferase from Salmonella typhimurium controls enzymic activity
The COOH terminus of arylamine N-acetyltransferase from Salmonella typhimurium controls enzymic activity
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DOI：
10.1074/jbc.m104365200
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发表时间：
2002-04-05
影响因子：
4.8
通讯作者：
Sim, E
中科院分区：
文献类型：
--
作者：
Mushtaq, A;Payton, M;Sim, E
芳胺 N-乙酰转移酶 (NAT) 是一个同源酶家族，可通过乙酰辅酶 A (Ac-CoA) 的乙酰基转移对芳胺、芳基羟胺和芳基肼进行乙酰化，存在于许多生物体中。 NAT 最初被确定为导致人类抗结核药物异烟肼失活的酶。鼠伤寒沙门氏菌 NAT 的三维结构已被解析，并显示其具有三个不同的结构域和一个由“Cys(69)-His(107)-Asp(122)”组成的活性位点催化三联体，这是半胱氨酸蛋白酶等水解酶的典型特征。晶体晶胞由四聚体的二聚体组成，各个单体的 C 末端并列。为了研究鼠伤寒沙门氏菌全长 NAT 的前两个结构域的功能并研究 NAT C 末端的作用，制备了 C 端十一肽或整个第三个结构域（85 个氨基酸）缺失的截短突变体。与全长 NAT 蛋白（281 个氨基酸）不同，来自鼠伤寒沙门氏菌的 NAT 截短突变体在大肠杆菌细胞内过表达时具有毒性。全长 NAT 只能在芳胺底物存在的情况下水解 Ac-CoA。然而，即使在没有芳基胺底物的情况下，两种截短突变体也会水解 Ac-CoA，这说明 C 端十一肽通过来自鼠伤寒沙门氏菌的 NAT 控制 Ac-CoA 的水解。
Arylamine N-acetyltransferases (NATs) are a homologous family of enzymes, which acetylate arylamines, arylhydroxylamines, and arylhydrazines by acetyl transfer from acetyl-coenzyme A (Ac-CoA) and are found in many organisms. NAT was first identified as the enzyme responsible for the inactivation of the anti-tubercular drug isoniazid in humans. The three-dimensional structure of NAT from Salmonella typhimurium has been resolved and shown to have three distinct domains and an active site catalytic triad composed of "Cys(69)-His(107)-Asp(122)," which is typical of hydrolytic enzymes such as the cysteine proteases. The crystal unit cell consists of a dimer of tetramers, with the C terminus of individual monomers juxtaposed. To investigate the function of the first two domains of full-length NAT from S. typhimurium and to investigate the role of the C terminus of NAT, truncation mutants were made with either the C-terminal undecapeptide or the entire third domain (85 amino acids) missing. Unlike the full-length NAT protein (281 amino acids), the truncation mutants of NAT from S. typhimurium are toxic when overexpressed intracellularly in Escherichia coli. Full-length NAT hydrolyses Ac-CoA but only in the presence of an arylamine substrate. Both truncation mutants, however, hydrolyze Ac-CoA even in the absence of arylamine substrate, illustrating that the C-terminal undecapeptide controls hydrolysis of Ac-CoA by NAT from S. typhimurium.