Structural differences of hydroxycinnamoyl transferases in Lamiaceae - Part 1: The structure of rosmarinic acid synthase

羟基肉桂酰基转移酶(HCT)是植物新陈代谢中的重要酶，本课题主要研究BAHD酰基转移酶超家族中的HCT，特别是在一个系统发育分支中。这些HCT将辅酶A中的羟基肉桂酸部分转移到受体底物，如莽草酸、奎宁酸酯或羟基苯乳酸盐，最终形成单木醇、绿原酸和迷迭香酸等化合物。参与这些生物合成的HCT显示出高度的同源性。尽管它们对植物新陈代谢很重要，但关于这些酶的结构信息很少，但迫切需要解释报道的HCT对不同底物的接受以及同一种酶形成酯和酰胺的原因。从进化的角度来看，HCT活性中心的变化导致不同的底物偏好是有趣的。在第一次应用期间，我们试图从Coleus blumei中结晶迷迭香酸合成酶(RAS)-不幸的是没有足够的成功。现在，我们想要运用我们在处理HCT方面的丰富经验，进行一种新的尝试，以深入了解HCT的结构。除了结晶作用外，还将使用建模和基片对接。该项目的更多部分是定点突变和构建不同HCT的嵌合蛋白，并随后测试它们的底物接受性。

Hydroxycinnamoyltransferases (HCTs) are important enzymes in plant metabolism, in this project we are interested in the HCTs from the BAHD acyltransferase superfamily, especially within one phylogenetic clade. These HCTs transfer a hydroxycinnamoyi moiety from coenzyme A to an acceptor substrate such as shikimate, quinate or hydroxyphenyllactate which finally results in the formation of compounds like monolignols, chlorogenic acid and rosmarinic acid, respectively. The HCTs involved in these biosyntheses show high homology. Despite their importance for plant metabolism, structural information on these enzymes is scarce, but it is urgently needed to explain the diverse substrate acceptance that was reported for HCTs as well as the formation of esters and amides by the same enzyme. From an evolutionary point of view, the changes in the active centres of HCTs resulting in different substrate preferences are interesting. During the first application period we have tried to crystallise rosmarinic acid synthase (RAS) from Coleus blumei - unfortunately without sufficient success. We now want to apply our ample experiences with handling HCTs for a novel attempt to get insight into the structure of HCTs. Modelling and substrate docking will be used besides crystallisation. Further parts of this project are site-directed mutagenesis and construction of chimeric proteins of different HCTs with subsequent testing of their substrate acceptance.