Active site residues governing substrate selectivity and polyketide chain length in aloesone synthase
Active site residues governing substrate selectivity and polyketide chain length in aloesone synthase
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DOI：
10.1111/j.1742-4658.2005.05059.x
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发表时间：
2006-01-01
期刊：
影响因子：
5.4
通讯作者：
Noguchi, H
中科院分区：
文献类型：
--
作者：
Abe, I;Watanabe, T;Noguchi, H
芦荟酮合成酶（ALS）和查尔酮合成酶（CHS）是植物特有的III型聚酮合成酶，它们具有62%的氨基酸序列同源性。ALS选择乙酰辅酶a作为起始物，与丙二酰辅酶a连续六次缩合生成七肽单酮，而CHS则催化4-丙二酰辅酶a与三个丙二酰辅酶a缩合生成查尔酮。在ALS中，CHS的Thr197、Gly256和Ser338，即沿起始/延伸腔排列的活性位点残基，分别被Ala、Leu和Thr唯一地取代。同源性模型预测，相对于CHS， ALS的活性位点结构结合了“水平限制”的G256L替换和“向下扩展”的T197A替换。此外，ALS有一个额外的埋藏袋，延伸到活动部位腔的“底部”。因此，空间调制有利于ALS利用较小的乙酰辅酶a起始物，同时为额外的聚酮链延伸提供足够的体积。事实上，这些位点（A197T、L256G和T338S）的类chs点突变完全破坏了七肽的产生活性。相反，A197T突变体产生一个五酮，2,7-二羟基-5-甲基色素，而L256G和T338S只产生一个三酮，三乙酸内酯。相比之下，L256G接受4-香豆酰辅酶a作为发酵剂，有效地产生了四肽，4-香豆酰三乙酸内酯。这些结果表明，Gly256决定了起始底物的选择性，而位于埋袋入口的Thr197控制了聚酮链的长度。最后，靠近催化Cys164的Ser338引导线性聚酮中间体延伸到囊中，从而导致掌大黄ALS肝肽的形成。
Aloesone synthase (ALS) and chalcone synthase (CHS) are plant-specific type III poyketide synthases sharing 62% amino acid sequence identity. ALS selects acetyl-CoA as a starter and carries out six successive condensations with malonyl-CoA to produce a heptaketide aloesone, whereas CHS catalyses condensations of 4-coumaroyl-CoA with three malonyl-CoAs to generate chalcone. In ALS, CHS's Thr197, Gly256, and Ser338, the active site residues lining the initiation/elongation cavity, are uniquely replaced with Ala, Leu, and Thr, respectively. A homology model predicted that the active site architecture of ALS combines a 'horizontally restricting' G256L substitution with a 'downward expanding' T197A replacement relative to CHS. Moreover, ALS has an additional buried pocket that extends into the 'floor' of the active site cavity. The steric modulation thus facilitates ALS to utilize the smaller acetyl-CoA starter while providing adequate volume for the additional polyketide chain extensions. In fact, it was demonstrated that CHS-like point mutations at these positions (A197T, L256G, and T338S) completely abolished the heptaketide producing activity. Instead, A197T mutant yielded a pentaketide, 2,7-dihydroxy-5-methylchromone, while L256G and T338S just afforded a triketide, triacetic acid lactone. In contrast, L256G accepted 4-coumaroyl-CoA as starter to efficiently produce a tetraketide, 4-coumaroyltriacetic acid lactone. These results suggested that Gly256 determines starter substrate selectivity, while Thr197 located at the entrance of the buried pocket controls polyketide chain length. Finally, Ser338 in proximity of the catalytic Cys164 guides the linear polyketide intermediate to extend into the pocket, thus leading to formation of the hepataketide in Rheum palmatum ALS.