体外重建和 6-脱氧赤藓醇 B 合酶的分析。
In vitro reconstitution and analysis of the 6-deoxyerythronolide B synthase.
机构信息
Department of Chemical Engineering, ‡Department of Chemistry, §School of Medicine, and ⊥Medical Science Training Program, Stanford University , Stanford, California 94305, United States.
出版信息
J Am Chem Soc. 2013 Nov 13;135(45):16809-12. doi: 10.1021/ja409048k. Epub 2013 Nov 1.
Notwithstanding an extensive literature on assembly line polyketide synthases such as the 6-deoxyerythronolide B synthase (DEBS), a complete naturally occurring synthase has never been reconstituted in vitro from purified protein components. Here, we describe the fully reconstituted DEBS and quantitatively characterize some of the properties of the assembled system that have never been explored previously. The maximum turnover rate of the complete hexamodular system is 1.1 min(-1), comparable to the turnover rate of a truncated trimodular derivative (2.5 min(-1)) but slower than that of a bimodular derivative (21 min(-1)). In the presence of similar concentrations of methylmalonyl- and ethylmalonyl-CoA substrates, DEBS synthesizes multiple regiospecifically modified analogues, one of which we have analyzed in detail. Our studies lay the foundation for biochemically interrogating and rationally engineering polyketide assembly lines in an unprecedented manner.
尽管有大量关于装配线聚酮合酶的文献，如 6-脱氧红霉内酯 B 合酶（DEBS），但从未从纯化的蛋白质成分中重新构建出完整的天然存在的合酶。在这里，我们描述了完全重组的 DEBS，并定量表征了以前从未探索过的组装系统的一些特性。完整的六模块系统的最大周转率为 1.1 min(-1)，与截短的三模块衍生物（2.5 min(-1)）的周转率相当，但比双模块衍生物（21 min(-1)）慢。在类似浓度的甲基丙二酰-CoA 和乙基丙二酰-CoA 底物存在的情况下，DEBS 合成了多种具有区域特异性的修饰类似物，其中一种我们已经进行了详细分析。我们的研究为以前所未有的方式对聚酮装配线进行生物化学研究和合理工程化奠定了基础。
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