State Key Laboratory of Phytochemistry and Plant Resources in West China, and CAS Center for Excellence in Molecular Plant Sciences, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China.
The Key Laboratory of Synthetic Biology, and CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China.
Nat Commun. 2019 Sep 6;10(1):4036. doi: 10.1038/s41467-019-11987-z.
The skeleton of tropane alkaloids is derived from ornithine-derived N-methylpyrrolinium and two malonyl-CoA units. The enzymatic mechanism that connects N-methylpyrrolinium and malonyl-CoA units remains unknown. Here, we report the characterization of three pyrrolidine ketide synthases (PYKS), AaPYKS, DsPYKS, and AbPYKS, from three different hyoscyamine- and scopolamine-producing plants. By examining the crystal structure and biochemical activity of AaPYKS, we show that the reaction mechanism involves PYKS-mediated malonyl-CoA condensation to generate a 3-oxo-glutaric acid intermediate that can undergo non-enzymatic Mannich-like condensation with N-methylpyrrolinium to yield the racemic 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This study therefore provides a long sought-after biosynthetic mechanism to explain condensation between N-methylpyrrolinium and acetate units and, more importantly, identifies an unusual plant type III polyketide synthase that can only catalyze one round of malonyl-CoA condensation.
托烷生物碱的骨架来源于鸟氨酸衍生的 N-甲基吡咯啉鎓和两个丙二酰辅酶 A 单元。将 N-甲基吡咯啉鎓和丙二酰辅酶 A 单元连接起来的酶促机制尚不清楚。在这里，我们报告了来自三种不同的莨菪碱和东莨菪碱产生植物的三种吡咯烷酮酮合酶 (PYKS)，即 AaPYKS、DsPYKS 和 AbPYKS 的特性。通过检查 AaPYKS 的晶体结构和生化活性，我们表明反应机制涉及 PYKS 介导的丙二酰辅酶 A 缩合，生成 3-氧代戊二酸中间产物，该中间产物可以与 N-甲基吡咯啉鎓进行非酶促类似 Mannich 缩合，生成外消旋 4-(1-甲基-2-吡咯烷基)-3-氧代丁酸。因此，这项研究提供了一个长期以来被寻求的生物合成机制，解释了 N-甲基吡咯啉鎓和乙酸盐单元之间的缩合，更重要的是，鉴定了一种不寻常的植物类型 III 聚酮合酶，它只能催化一轮丙二酰辅酶 A 缩合。
