Mao Guotao, Zhao Yan, Kang Xusheng, Li Zhijie, Zhang Yan, Wang Xianping, Sun Fei, Sankaran Krishnan, Zhang Xuejun C
National Laboratory of Macromolecules, National Center of Protein Science - Beijing, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Nat Commun. 2016 Jan 5;7:10198. doi: 10.1038/ncomms10198.
Lipoprotein biogenesis is essential for bacterial survival. Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is an integral membrane enzyme that catalyses the first reaction of the three-step post-translational lipid modification. Deletion of the lgt gene is lethal to most Gram-negative bacteria. Here we present the crystal structures of Escherichia coli Lgt in complex with phosphatidylglycerol and the inhibitor palmitic acid at 1.9 and 1.6 Å resolution, respectively. The structures reveal the presence of two binding sites and support the previously reported structure-function relationships of Lgt. Complementation results of lgt-knockout cells with different mutant Lgt variants revealed critical residues, including Arg143 and Arg239, that are essential for diacylglyceryl transfer. Using a GFP-based in vitro assay, we correlated the activities of Lgt with structural observations. Together, the structural and biochemical data support a mechanism whereby substrate and product, lipid-modified lipobox-containing peptide, enter and leave the enzyme laterally relative to the lipid bilayer.
脂蛋白生物合成对细菌存活至关重要。磷脂酰甘油：前脂蛋白二酰甘油转移酶（Lgt）是一种整合膜酶，催化三步翻译后脂质修饰的第一步反应。lgt基因的缺失对大多数革兰氏阴性细菌是致命的。在此，我们分别展示了分辨率为1.9 Å和1.6 Å的与磷脂酰甘油和抑制剂棕榈酸结合的大肠杆菌Lgt的晶体结构。这些结构揭示了两个结合位点的存在，并支持先前报道的Lgt的结构-功能关系。用不同突变的Lgt变体对lgt基因敲除细胞进行互补实验，揭示了关键残基，包括Arg143和Arg239，它们对二酰甘油转移至关重要。使用基于绿色荧光蛋白（GFP）的体外检测方法，我们将Lgt的活性与结构观察结果相关联。总之，结构和生化数据支持一种机制，即底物和产物，即脂质修饰的含脂盒肽，相对于脂质双层横向进出酶。
