辅酶Q生物合成限速酶UbiG心磷脂特异性结合、底物识别及催化机制的核磁共振研究

辅酶Q是一种重要的脂质分子，在细胞呼吸链中发挥关键作用，也是体内天然的抗氧化剂。UbiG及其真核同源蛋白Coq3，属于SAM依赖的氧-甲基转移酶家族，是辅酶Q生物合成的限速酶，催化其中两步氧-甲基化反应。我们最近发现UbiG/Coq3是一类新的膜结合蛋白，能够特异性地识别心磷脂。然而，UbiG/Coq3心磷脂特异性识别的结构基础尚不清楚，其对膜上底物识别与催化的分子机制也不清楚。本申请拟，在现有工作的基础上，以大肠杆菌UbiG为研究对象，应用液体核磁共振和nanodiscs膜模拟体系，研究UbiG与心磷脂的相互作用，旨在揭示其心磷脂特异性结合的结构基础，并通过建立磷脂膜环境下的UbiG酶活测定方法，研究膜结合对其酶活的影响。同时，我们还将研究UbiG对膜上底物的识别，旨在了解其催化机制。研究结果将为深入理解UbiG/Coq3在辅酶Q生物合成途径中的工作机制奠定基础。

Coenzyme Q is an essential lipid that plays key roles in the cellular respiratory chain. It also severs as a natural antioxidant. UbiG and Coq3 (orthologue in eukaryotes) belong to the SAM-dependent methyltransferase family. They are rate-limiting enzymes that catalyse both O-methylation steps in CoQ biosynthesis. Recently, we have found that UbiG/Coq3 defines a class of novel-membrane binding proteins and are capable of binding cardiolipin. However, the structural basis that governs the specific cardiolipin-binding of UbiG/Coq3 is unclear. In addition, the mechanisms of its substrate recognition and catalyzation also remain unclear. In this proposal, we will study the recognition of cardiolipin by E.coli UbiG, employing NMR methods together with nanodiscs membrane mimic. In addition, we will develop new methods to measure enzymatic activity of UbiG under membrane environment and then test the effects of cardiolipin binding on its enzymatic activity. Furthermore, in order to understand the catalytic mechanisms of UbiG，we will also study its substrates recognition under lipid membrane environment. The results will shed lights on the functional mechanisms of UbiG/Coq3 in coenzyme Q biosynthesis pathway.