Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis
Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis
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DOI：
10.1073/pnas.0913531107
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发表时间：
2010-04-06
影响因子：
11.1
通讯作者：
Tsai, Shiou-Chuan
中科院分区：
文献类型：
--
作者：
Korman, Tyler Paz;Crawford, Jason M.;Tsai, Shiou-Chuan
聚酮化合物天然产物具有不同的结构和生物学功能，并与脂肪酸合成共享生物合成步骤的子集。由聚酮化合物脱氢酶（PKS）和脂肪酸脱氢酶催化的最终转化最常由硫酯酶（TE）进行。TE结构域在真菌非还原性迭代PKS（NR-PKS）中的合成多功能性已被证明通过催化C-C环闭合反应而扩展到克莱森环化酶（CLC）化学，这与先前报道的TE结构中观察到的硫酯水解或O-C/N-C大环化相反。催化C-C键形成作为产物释放机制极大地扩展了PKS的合成潜力，但这种活性是如何获得的仍然是一个谜。我们报告的TE/CLC结构域的聚酮合酶A，多域PKS的生物合成的黄曲霉毒素B-1，一个强大的环境致癌物的中心的生化和结构分析。诱变实验证实了预测的催化三联体的身份和它的作用，在催化最后的克莱森型环化的黄曲霉毒素前体，去甲异喹啉酸蒽酮。1.7埃的晶体结构显示了具有独特疏水底物结合室的催化封闭形式的α/β-水解酶折叠。我们建议，一个关键的旋转的基板侧链耦合到一个蛋白质的构象变化从开放到封闭的形式空间支配基板定位和C-C环化。生化研究，TE/CLC域的1.7埃晶体结构，和中间建模提供了第一个机制的见解，这种广泛分布的C-C键形成类的TE。
Polyketide natural products possess diverse architectures and biological functions and share a subset of biosynthetic steps with fatty acid synthesis. The final transformation catalyzed by both polyketide synthases (PKSs) and fatty acid synthases is most often carried out by a thioesterase (TE). The synthetic versatility of TE domains in fungal nonreducing, iterative PKSs (NR-PKSs) has been shown to extend to Claisen cyclase (CLC) chemistry by catalyzing C-C ring closure reactions as opposed to thioester hydrolysis or O-C/N-C macrocyclization observed in previously reported TE structures. Catalysis of C-C bond formation as a product release mechanism dramatically expands the synthetic potential of PKSs, but how this activity was acquired has remained a mystery. We report the biochemical and structural analyses of the TE/CLC domain in polyketide synthase A, the multidomain PKS central to the biosynthesis of aflatoxin B-1, a potent environmental carcinogen. Mutagenesis experiments confirm the predicted identity of the catalytic triad and its role in catalyzing the final Claisen-type cyclization to the aflatoxin precursor, norsolorinic acid anthrone. The 1.7 angstrom crystal structure displays an alpha/beta-hydrolase fold in the catalytic closed form with a distinct hydrophobic substrate-binding chamber. We propose that a key rotation of the substrate side chain coupled to a protein conformational change from the open to closed form spatially governs substrate positioning and C-C cyclization. The biochemical studies, the 1.7 angstrom crystal structure of the TE/CLC domain, and intermediate modeling afford the first mechanistic insights into this widely distributed C-C bond-forming class of TEs.