Structural Insight into the Reaction Mechanism of Ketosynthase-Like Decarboxylase in a Loading Module of Modular Polyketide Synthases
Structural Insight into the Reaction Mechanism of Ketosynthase-Like Decarboxylase in a Loading Module of Modular Polyketide Synthases
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DOI：
10.1021/acschembio.1c00856
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发表时间：
2022-01-05
影响因子：
4
通讯作者：
Eguchi, Tadashi
中科院分区：
文献类型：
--
作者：
Chisuga, Taichi;Nagai, Akira;Eguchi, Tadashi
酮酶样脱羧酶（KSQ）结构域广泛分布于模块化聚酮酶（PKS）的装载模块中，并被提出催化丙二酰或甲基丙二酰单元的脱羧以构建PKS起始单元。KSQ结构域与酮合酶（ketosynthase，KS）结构域具有很高的序列相似性，除了KS结构域的催化Cys残基在KSQ结构域中被Gln取代外，KS结构域在聚酮化合物和脂肪酸生物合成中催化转酰和脱羧缩合。在这里，我们提出了GfsA KSQ和CmiP 4 KSQ的生化分析，它们分别参与FD-891和克雷霉素的生物合成。体外分析表明，这些KSQ结构域催化丙二酰和甲基丙二酰单元的脱羧。此外，我们确定了GfsA KSQ与丙二酰硫酯底物类似物的复合物的晶体结构，这使得能够鉴定脱羧反应中涉及的关键氨基酸残基。突变分析证实了这些残基的重要性。基于这些发现，我们提出了一种机制的GfsA KSQ催化的脱羧反应。GfsA KSQ通过将底物固定在适合脱羧的构象中来启动脱羧。脱羧后烯醇化物的形成由两个保守的苏氨酸残基辅助。比较GfsA KSQ与KS结构域的结构表明，KSQ结构域活性位点中的Gln残基模拟KS结构域活性位点中的酰化Cys残基。
Ketosynthase-like decarboxylase (KSQ) domains are widely distributed in the loading modules of modular polyketide synthases (PKSs) and are proposed to catalyze the decarboxylation of a malonyl or methylmalonyl unit for the construction of the PKS starter unit. KSQ domains have high sequence similarity to ketosynthase (KS) domains, which catalyze transacylation and decarboxylative condensation in polyketide and fatty acid biosynthesis, except that the catalytic Cys residue of KS domains is replaced by Gln in KSQ domains. Here, we present biochemical analyses of GfsA KSQ and CmiP4 KSQ, which are involved in the biosynthesis of FD-891 and cremimycin, respectively. In vitro analysis showed that these KSQ domains catalyze the decarboxylation of malonyl and methylmalonyl units. Furthermore, we determined the crystal structure of GfsA KSQ in complex with a malonyl thioester substrate analogue, which enabled identification of key amino acid residues involved in the decarboxylation reaction. The importance of these residues was confirmed by mutational analysis. On the basis of these findings, we propose a mechanism of the decarboxylation reaction catalyzed by GfsA KSQ. GfsA KSQ initiates decarboxylation by fixing the substrate in a suitable conformation for decarboxylation. The formation of enolate upon decarboxylation is assisted by two conserved threonine residues. Comparison of the structure of GfsA KSQ with those of KS domains suggests that the Gln residue in the active site of the KSQ domain mimics the acylated Cys residue in the active site of KS domains.