Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units
Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units
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DOI：
10.1021/bi9820311
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发表时间：
1999-02-02
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：
Lau, J;Fu, H;Khosla, C
模块化聚酮化合物合酶（PKS），例如6-脱氧赤霉酸内酯B合酶（DEBS），是组织成模块的大型多功能酶复合物，其中每个模块都携带催化延伸单元缩合到生长的聚酮化合物链上所需的结构域。每个模块还决定了在链延长过程中引入主链的手性中心的立体化学。在这里，我们使用域诱变研究的作用，酰基转移酶（AT）域的个别模块的选择和立体化学的命运的扩展单元。我们的研究结果表明，AT结构域的DEBS不影响差向异构化的（2S）-甲基丙二酰辅酶A扩展单元。因此，DEBS产生的甲基分支中心的立体化学控制最有可能驻留在各个模块的酮合酶（KS）结构域。相比之下，最近的几项研究表明，延伸单位的特异性可以改变AT结构域取代。在这些实施例中的一些中，所得聚酮化合物以比相应的天然产物低得多的滴度产生。我们分析了一个这样的减毒突变的DEBS，其中甲基丙二酰转移酶结构域的模块2被替换为丙二酰转移酶结构域。如前所述，所得PKS仅产生少量预期的6-脱氧阿糖胞苷B的去甲基类似物。然而，当相同的混合模块作为末端模块放置在截短的2-模块PKS中时，它产生了几乎正常量的预期去甲基三酮内酯。这些结果说明了这些多功能酶的模块化的限制。为了剖析特定氨基酸在控制AT底物特异性中的作用，我们在选定的丙二酰转移酶和甲基丙二酰转移酶之间交换了几段氨基酸，发现所有AT结构域中存在的短（23-35个氨基酸）C-末端片段是其底物特异性的主要决定因素。有趣的是，它的长度和氨基酸序列在已知的AT结构域之间变化很大。因此，我们建议，选择的PKS模块的扩展单元的影响，由一个“高变区”，这可以通过操纵组合诱变产生新的AT结构域具有放松或改变底物特异性。
Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzyme complexes that are organized into modules, where each module carries the domains needed to catalyze the condensation of an extender unit onto a growing polyketide chain. Each module also dictates the stereochemistry of the chiral centers introduced into the backbone during the chain elongation process. Here we used domain mutagenesis to investigate the role of the acyl transferase (AT) domains of individual modules in the choice and stereochemical fate of extender units. Our results indicate that the AT domains of DEBS do not influence epimerization of the (2S)-methylmalonyl-CoA extender units. Hence, stereochemical control of the methyl-branched centers generated by DEBS most likely resides in the ketosynthase (KS) domains of the individual modules. In contrast, several recent studies have demonstrated that extender unit specificity can be altered by AT domain substitution. In some of these examples, the resulting polyketide was produced at considerably lower titers than the corresponding natural product. We analyzed one such attenuated mutant of DEBS, in which the methylmalonyl transferase domain of module 2 was replaced with a malonyl transferase domain. As reported earlier, the resulting PKS produced only small quantities of the expected desmethyl analogue of 6-deoxyerythronolide B. However, when the same hybrid module was placed as the terminal module in a truncated 2-module PKS, it produced nearly normal quantities of the expected desmethyl triketide lactone. These results illustrate the limits to modularity of these multifunctional enzymes. To dissect the role of specific amino acids in controlling AT substrate specificity, we exchanged several segments of amino acids between selected malonyl and methylmalonyl transferases, and found that a short (23-35 amino acid) C-terminal segment present in all AT domains is the principal determinant of their substrate specificity. Interestingly, its length and amino acid sequence vary considerably among the known AT domains. We therefore suggest that the choice of extender units by the PKS modules is influenced by a "hypervariable region", which could be manipulated via combinatorial mutagenesis to generate novel AT domains possessing relaxed or altered substrate specificity.