Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa
Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa
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DOI：
10.1073/pnas.1011499107
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发表时间：
2010-11-16
影响因子：
11.1
通讯作者：
Abe, Ikuro
中科院分区：
文献类型：
--
作者：
Morita, Hiroyuki;Wanibuchi, Kiyofumi;Abe, Ikuro
来自Oryza sativa的Curcuminoid synthase （CUS）是一种植物特异性的III型聚酮合成酶（PKS），它催化双去甲氧基姜黄素的C(6)-C(7)-C(6)二芳基七烷类支架，通过两个4- coumaryl - coa分子和一个丙二酰coa分子的缩合而成。在2.5埃的分辨率下分析了O. sativa CUS的晶体结构，揭示了一个独特的向下扩展的活性位点结构，这是以前在已知的III型pks中未发现的。大的活性位腔足够长，可以容纳两个C(6)-C(3)香豆基单元和一个丙二烯基单元。此外，晶体结构表明存在一个假定的亲核水分子，该分子与Ser351-Asn142-H(2)O-Tyr207-Glu202形成氢键网络，在活性位点中心邻近催化Cys174。这些观察结果表明，CUS采用独特的催化机制一锅形成C(6)-C(7)-C(6)支架。因此，CUS利用亲核水在双酮阶段终止初始聚酮链延伸。酶结合中间体的硫酯键裂解生成4-香豆酰二酮酸，然后将其保存在向下扩展的口袋中，随后与第二个4-香豆酰辅酶a起始物进行脱羧缩合，生成双去甲氧基姜黄素。基于结构的位点导向突变体M265L和G274F改变了底物和产物的特异性，接受4-羟基苯基丙炔辅酶a作为起始物产生四氢双去甲氧基姜黄素。这些发现不仅为CUS的催化机制提供了结构基础，而且为扩大III型PKS酶的生物合成库提供了进一步的策略。
Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C(6)-C(7)-C(6) diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5-angstrom resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C(6)-C(3) coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H(2)O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C(6)-C(7)-C(6) scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin. The structure-based site-directed mutants, M265L and G274F, altered the substrate and product specificities to accept 4-hydroxyphenylpropionyl-CoA as the starter to produce tetrahydrobisdemethoxycurcumin. These findings not only provide a structural basis for the catalytic machinery of CUS but also suggest further strategies toward expanding the biosynthetic repertoire of the type III PKS enzymes.