Biosynthesis of 3-hydroxybenzoate-derived polyketides in Gentianaceae

龙胆科植物包括著名的药用植物，如红花半夏和紫花蛇牙菜。它们的成分包括口恶酮，其骨架是由一种称为二苯甲酮合成酶(BPS)的III型聚酮合成酶形成的。与金丝桃科的BPSS以苯甲酰辅酶A为起始底物不同，龙胆科的酶更倾向于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。苯基吡喃酮合成酶(PPS)也是如此，它的编码活性和非活性异构体的cDNA已经被克隆。这些同工酶只有四种位于表面的氨基酸不同。突变失活亚型中的每一个残基都会产生活性酶蛋白。BPSs和PPSs都将从生化和结构性质方面进行表征。结晶和结构分析将与BlankenFeldt教授小组(布伦斯韦格Helmholtz感染研究中心)合作进行。为了解决3-(3-羟基苯甲酰氧基)苯甲酰辅酶A的生理意义，将采用基于聚合酶链式反应的克隆和转录组挖掘的方法来检测和分离3-羟基苯甲酰转移酶。

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.