Biosynthesis of 3-hydroxybenzoate-derived polyketides in Gentianaceae

龙胆科包括著名的药用植物，如半人马座和獐牙菜。它们的成分包括山酮，其骨架由III型聚酮合成酶形成，称为二苯甲酮合成酶（BPS）。与金丝桃科的bps使用苯甲酰辅酶a作为起始底物相比，龙胆科的酶更倾向于使用3-羟基化的起始单位。最近观察到，含有两个3-羟基苯甲酰残基的辅酶a酯，即3-（3-羟基苯甲酰氧基）苯甲酰辅酶a甚至是最好的底物。对于苯丙酮合成酶（PPS）也是如此，其编码活性和非活性异构体的cdna已被克隆。这些同工酶只在表面有四个氨基酸不同。对失活异构体中的每一个残基进行诱变产生活性酶蛋白。bps和PPSs都将从生化和结构性质上进行表征。结晶和结构分析将与Blankenfeldt教授小组（亥姆霍兹感染研究中心，布伦瑞克）合作进行。为了研究3-（3-羟基苯甲酰氧基）苯甲酰辅酶a的生理相关性，我们将利用基于pcr的克隆和转录组挖掘技术，对3-羟基苯甲酰转移酶进行检测和cDNA分离。

The Gentianaceae include well-known medicinal plants, such as Centaurium erythraea and Swertia chirata. Their profile of constituents involves xanthones, the skeleton of which is formed by a type III polyketide synthase, called benzophenone synthase (BPS). In contrast to BPSs from Hypericaceae, which use benzoyl-CoA as starter substrate, the enzymes from Gentianaceae prefer the 3-hydroxylated starter unit. As recently observed, the CoA ester with two 3-hydroxybenzoyl residues, i.e. 3-(3-hydroxybenzoyloxy)benzoyl-CoA is even the best substrate. This is also true for phenylpyrone synthase (PPS), for which cDNAs encoding both an active and an inactive isoform have been cloned. These isoenzymes differ in only four amino acids located at the surface. Mutagenesis of each of these residues in the inactive isoform yields active enzyme protein. Both BPSs and PPSs will be characterized with respect to biochemical and structural properties. Crystallization and structure analysis will be carried out in cooperation with the Prof. Blankenfeldt group (Helmholtz Center for Infection Research, Braunschweig). To address the physiological relevance of 3-(3-hydroxybenzoyloxy)benzoyl-CoA, detection and cDNA isolation of 3-hydroxybenzoyl transferase will be tackled, using PCR-based cloning and transcriptome mining.