6-甲基水杨酸合酶催化结构域的产物释放的隐藏功能。
Hidden function of catalytic domain in 6-methylsalicylic acid synthase for product release.
机构信息
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033.
School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694, Japan.
出版信息
J Biol Chem. 2010 May 14;285(20):15637-15643. doi: 10.1074/jbc.M110.107391. Epub 2010 Mar 19.
Functional investigation of the proposed dehydratase domain of ATX, a 6-methylsalicylic acid synthase from Aspergillus terreus, revealed that the domain is not involved in dehydration of the beta-hydroxytriketide intermediate tethered on the acyl carrier protein but catalyzes thioester hydrolysis to release the product from the acyl carrier protein. Thus, we renamed this domain the thioester hydrolase (TH) domain. The intermediate bound to the TH domain of mutant H972A formed in the presence of NADPH was released as 6-methylsalicylic acid by both the intact ATX and by THID (a 541-amino acid region containing TH domain and its downstream) protein, in trans. Furthermore, THID showed a catalytic activity to hydrolyze a model substrate, 6-methylsalicylic acid-N-acetylcysteamine. The TH domain is the first example of a product-releasing domain that is located in the middle of a multidomain iterative type I polyketide synthase. Moreover, it is functionally different from serine protease-type thioesterase domains of iterative type I polyketide synthases.
功能研究表明，来自土曲霉的 6-甲基水杨酸合酶 ATX 的脱水酶结构域不参与β-羟三酮中间产物与酰基载体蛋白的脱水，而是催化硫酯水解，将产物从酰基载体蛋白上释放出来。因此，我们将该结构域重新命名为硫酯水解酶（TH）结构域。在 NADPH 存在的情况下，突变体 H972A 中形成的与 TH 结构域结合的中间产物，无论是完整的 ATX 还是 THID（一种含有 TH 结构域及其下游的 541 个氨基酸区域）蛋白，都能以反式方式释放出 6-甲基水杨酸。此外，THID 还表现出催化水解模型底物 6-甲基水杨酸-N-乙酰半胱氨酸的活性。TH 结构域是第一个位于多结构域迭代型 I 聚酮合酶中间的产物释放结构域。此外，它在功能上与迭代型 I 聚酮合酶的丝氨酸蛋白酶型硫酯酶结构域不同。
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