Mechanistic analysis of a type II polyketide synthase.: Role of conserved residues in the β-ketoacyl synthase-chain length factor heterodimer
Mechanistic analysis of a type II polyketide synthase.: Role of conserved residues in the β-ketoacyl synthase-chain length factor heterodimer
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DOI：
10.1021/bi992121l
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发表时间：
2000-02-29
期刊：
影响因子：
2.9
通讯作者：
Khosla, C
中科院分区：
文献类型：
--
作者：
Dreier, J;Khosla, C
II型聚酮酶（PKS）是催化多功能芳香族天然产物（如放线菌紫素、弗伦诺菌素、四环霉素和多柔比星）的生物合成的多酶系统家族。这些系统中的每一个的中心组分是β-酮脂酰辅酶A酶-链长因子（KS-CLF）异二聚体。在酰基载体蛋白（ACP）和丙二酰辅酶A：ACP丙二酰转移酶（MAT）的存在下，该酶从丙二酰辅酶A合成确定长度的聚酮链。我们已经研究了放线菌紫素KS-CLF在引发，延长和终止其octaketide产品的作用，通过对野生型酶和选定的突变体进行检测，探测在整个催化循环的关键步骤。在反映PKS稳态营业额的条件下，检测到一种独特的酰基-ACP中间体，其携带长的，可能全长的酰基链。该物种不能由KS的C169 S、H309 A、K341 A和H346 A突变体合成，所有这些突变体都不能合成。在PKS催化循环的早期阶段被阻断。这四个残基在所有已知的KS中是普遍保守的。单独的丙二酰-ACP足以通过野生型KS-CLF在动力学和化学计量上有效地合成聚酮，但不能通过携带上述突变的异二聚体合成。在这些突变体中，C169 S是丙二酰-ACP的有效脱羧酶，但H309 A、K341 A和H346 A突变体不能催化脱羧。对于野生型酶以及C169 S和K341 A突变体，可以检测到标记从[C-14]丙二酰-ACP转移到KS中位置169处的亲核体，但对于H309 A突变体不能检测到，并且对于H346 A突变体仅非常弱。提出了一个模型的脱羧引发和延长的聚酮链的KS，其中C169和H346形成一个催化二联体的酰基链连接，H309的位置的丙二酰-ACP的活性位点，并支持负碳离子的形成，通过与硫酯羰基相互作用，和K341增强丙二酰-ACP脱羧率通过静电相互作用。我们的数据还表明，ACP和KS在每个C-C键形成事件后解离，并且新延伸的酰基链在解离发生之前从ACP泛酰巯基乙胺转移回KS半胱氨酸。链终止最可能是聚酮生物合成中的限速步骤。在act CLF中，与酮合酶的活性位点半胱氨酸对齐的普遍保守的S145残基和Q171都不是PKS活性所必需的。这里描述的结果提供了一个更好地了解II型PKS和脂肪酸脱氢酶的催化循环的基础。
Type II polyketide synthases (PKSs) are a family of multienzyme systems that catalyze the biosynthesis of polyfunctional aromatic natural products such as actinorhodin, frenolicin, tetracenomycin, and doxorubicin. A central component in each of these systems is the beta-ketoacyl synthase-chain length factor (KS-CLF) heterodimer. In the presence of an acyl carrier protein (ACP) and a malonyl-CoA:ACP malonyl transferase (MAT), this enzyme synthesizes a polyketide chain of defined length from malonyl-CoA. We have investigated the role of the actinorhodin KS-CLF in priming, elongation, and termination of its octaketide product by subjecting the wild-type enzyme and selected mutants to assays that probe key steps in the overall catalytic cycle. Under conditions reflecting steady-state turnover of the PKS, a unique acyl-ACP intermediate is detected that carries a long, possibly full-length, acyl chain. This species cannot be synthesized by the C169S, H309A, K341A, and H346A mutants of the KS, all of which. are blocked in early steps in the PKS catalytic cycle. These four residues are universally conserved in all known KSs. Malonyl-ACP alone is sufficient for kinetically and stoichiometrically efficient synthesis of polyketides by the wild-type KS-CLF, but not by heterodimers that carry the mutations listed above, Among these mutants, C169S is an efficient decarboxylase of malonyl-ACP, but the H309A, K341A, and H346A mutants are unable to catalyze decarboxylation. Transfer of label from [C-14]malonyl-ACP to the nucleophile at position 169 in the KS can be detected for the wild-type enzyme and for the C169S and K341A mutants, but not for the H309A mutant and only very weakly for the H346A mutant. A model is proposed for decarboxylative priming and extension of a polyketide chain by the KS, where C169 and H346 form a catalytic dyad for acyl chain attachment, H309 positions the malonyl-ACP in the active site and supports carbanion formation by interacting with the thioester carbonyl, and K341 enhances the rate of malonyl-ACP decarboxylation via electrostatic interaction. Our data also suggest that the ACP and the KS dissociate after each C-C bond forming event, and that the newly extended acyl chain is transferred back from the ACP pantetheine to the KS cysteine before dissociation can occur. Chain termination is most likely the rate-limiting step in polyketide biosynthesis. Within the act CLF, neither the universally conserved S145 residue nor Q171, which aligns with the active site cysteine of the ketosynthase, is essential for PKS activity. The results described here provide a basis for a better understanding of the catalytic cycle of type II PKSs and fatty acid synthases.