Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, California 90095, USA.
J Am Chem Soc. 2010 Apr 7;132(13):4530-1. doi: 10.1021/ja100060k.
Hypothemycin is a macrolide protein kinase inhibitor from the fungus Hypomyces subiculosus. During biosynthesis, its carbon framework is assembled by two iterative polyketide synthases (PKSs), Hpm8 (highly reducing) and Hpm3 (nonreducing). These were heterologously expressed in Saccharomyces cerevisiae BJ5464-NpgA, purified to near homogeneity, and reconstituted in vitro to produce (6'S,10'S)-trans-7',8'-dehydrozearalenol (1) from malonyl-CoA and NADPH. The structure of 1 was determined by X-ray crystallographic analysis. In the absence of functional Hpm3, the reducing PKS Hpm8 produces and offloads truncated pyrone products instead of the expected hexaketide. The nonreducing Hpm3 is able to accept an N-acetylcysteamine thioester of a correctly functionalized hexaketide to form 1, but it is unable to initiate polyketide formation from malonyl-CoA. We show that the starter-unit:ACP transacylase (SAT) of Hpm3 is critical for crosstalk between the two enzymes and that the rate of biosynthesis of 1 is determined by the rate of hexaketide formation by Hpm8.
Hypothemycin 是一种来自 Hypomyces subiculosus 的大环内酯蛋白激酶抑制剂。在生物合成过程中，其碳骨架由两个迭代的聚酮合酶（PKS）Hpm8（高度还原）和 Hpm3（非还原）组装而成。这些酶在酿酒酵母 BJ5464-NpgA 中异源表达，纯化至近均一性，并在体外重建以从丙二酰辅酶 A 和 NADPH 产生（6'S，10'S）-反式-7'，8'-脱氢玉米赤烯醇（1）。通过 X 射线晶体学分析确定了 1 的结构。在缺乏功能性 Hpm3 的情况下，还原 PKS Hpm8 会产生并卸载截断的吡喃酮产物，而不是预期的六酮。非还原的 Hpm3 能够接受正确官能化的六酮的 N-乙酰半胱氨酸硫酯以形成 1，但它无法从丙二酰辅酶 A 起始聚酮形成。我们表明，Hpm3 的起始单元：ACP 转酰基酶（SAT）对于两种酶之间的串扰至关重要，并且 1 的生物合成速率由 Hpm8 形成六酮的速率决定。
