Gu Liangcai, Geders Todd W, Wang Bo, Gerwick William H, Håkansson Kristina, Smith Janet L, Sherman David H
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA.
Science. 2007 Nov 9;318(5852):970-4. doi: 10.1126/science.1148790.
An unexpected biochemical strategy for chain initiation is described for the loading module of the polyketide synthase of curacin A, an anticancer lead derived from the marine cyanobacterium Lyngbya majuscula. A central GCN5-related N-acetyltransferase (GNAT) domain bears bifunctional decarboxylase/S-acetyltransferase activity, both unprecedented for the GNAT superfamily. A CurA loading tridomain, consisting of an adaptor domain, the GNAT domain, and an acyl carrier protein, was assessed biochemically, revealing that a domain showing homology to GNAT (GNAT(L)) catalyzes (i) decarboxylation of malonyl-coenzyme A (malonyl-CoA) to acetyl-CoA and (ii) direct S-acetyl transfer from acetyl-CoA to load an adjacent acyl carrier protein domain (ACP(L)). Moreover, the N-terminal adapter domain was shown to facilitate acetyl-group transfer. Crystal structures of GNAT(L) were solved at 1.95 angstroms (ligand-free form) and 2.75 angstroms (acyl-CoA complex), showing distinct substrate tunnels for acyl-CoA and holo-ACP(L) binding. Modeling and site-directed mutagenesis experiments demonstrated that histidine-389 and threonine-355, at the convergence of the CoA and ACP tunnels, participate in malonyl-CoA decarboxylation but not in acetyl-group transfer. Decarboxylation precedes acetyl-group transfer, leading to acetyl-ACP(L) as the key curacin A starter unit.
人们描述了一种用于抗癌先导化合物curacin A（一种源自海洋蓝藻Lyngbya majuscula的物质）聚酮合酶装载模块的意外生化链起始策略。一个核心的与GCN5相关的N - 乙酰转移酶（GNAT）结构域具有双功能脱羧酶/S - 乙酰转移酶活性，这对于GNAT超家族来说都是前所未有的。对由一个衔接子结构域、GNAT结构域和一个酰基载体蛋白组成的CurA装载三结构域进行了生化评估，结果表明一个与GNAT具有同源性的结构域（GNAT(L)）催化：（i）丙二酰辅酶A（丙二酰 - CoA）脱羧生成乙酰辅酶A，以及（ii）乙酰辅酶A直接进行S - 乙酰转移以装载相邻的酰基载体蛋白结构域（ACP(L)）。此外，N端衔接子结构域被证明有助于乙酰基转移。在1.95埃（无配体形式）和2.75埃（酰基辅酶A复合物）的分辨率下解析了GNAT(L)的晶体结构，显示出用于酰基辅酶A和全载ACP(L)结合的不同底物通道。建模和定点诱变实验表明，在辅酶A和ACP通道交汇处的组氨酸 - 389和苏氨酸 - 355参与丙二酰辅酶A的脱羧反应，但不参与乙酰基转移。脱羧反应先于乙酰基转移，导致乙酰 - ACP(L)成为curacin A关键的起始单元。
