School of Chemistry & Chemical Engineering , Queen's University Belfast , Belfast BT9 5AG , Northern Ireland , United Kingdom.
Department of Biocatalysis and Isotope Chemistry , Almac Sciences , Almac House, 20 Seagoe Industrial Estate , Craigavon BT63 5QD , Northern Ireland , United Kingdom.
J Phys Chem B. 2019 Aug 22;123(33):7092-7102. doi: 10.1021/acs.jpcb.9b04227. Epub 2019 Aug 7.
Lipoproteins are essential for bacterial survival. Bacterial lipoprotein biosynthesis is accomplished by sequential modification by three enzymes in the inner membrane, all of which are emerging antimicrobial targets. The X-ray crystal structure of prolipoprotein diacylglyceryl transferase (Lgt) and apolipoprotein -acyl transferase (Lnt) has been reported. However, the mechanisms of the post-translational modification catalyzed by these enzymes have not been understood. Here, we studied the mechanism of the transacylation reaction catalyzed by Lgt, the first enzyme for lipoprotein modification using molecular docking, molecular dynamics, and quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that Arg143, Arg239, and Glu202 play a critical role in stabilizing the glycerol-1-phosphate head group and activating the glycerol C3-O ester bond of the phosphatidylglycerol (PG) substrate. With PG binding, the opening of the L6-7 loop mediated by the highly conserved Arg236 residue as a gatekeeper is observed, which facilitates the release of the modified lipoprotein product, as well as the entry of another PG substrate. Further QM/MM studies revealed that His103 acts as a catalytic base to abstract a proton from the cysteine residue of the preproliprotein, initiating the diacylglyceryl transfer from PG to preprolipoprotein. This is the first study on the mechanism of lipoprotein modification catalyzed by a post-translocational processing enzyme. The transacylation mechanism of Lgt would shed light on the development of novel antimicrobial therapies targeting the challenging enzymes involved in the post-translocational modification pathway of lipoproteins.
脂蛋白对于细菌的生存至关重要。细菌脂蛋白的生物合成是通过内膜中的三种酶依次修饰完成的，这三种酶都是新兴的抗菌靶标。原脂蛋白二酰甘油转移酶（Lgt）和载脂蛋白酰基转移酶（Lnt）的 X 射线晶体结构已经报道。然而，这些酶催化的翻译后修饰的机制尚未被理解。在这里，我们使用分子对接、分子动力学和量子力学/分子力学（QM/MM）计算研究了 Lgt（脂蛋白修饰的第一酶）催化的转酰基反应的机制。我们的结果表明，Arg143、Arg239 和 Glu202 在稳定甘油-1-磷酸头基和激活磷脂酰甘油（PG）底物的甘油 C3-O 酯键方面发挥着关键作用。随着 PG 的结合，观察到由高度保守的 Arg236 残基作为门控分子介导的 L6-7 环的打开，这促进了修饰的脂蛋白产物的释放，以及另一个 PG 底物的进入。进一步的 QM/MM 研究表明，His103 作为一个催化碱，从原脂蛋白的半胱氨酸残基上夺取一个质子，从而启动从 PG 到原脂蛋白的二酰甘油转移。这是首次对翻译后加工酶催化的脂蛋白修饰机制进行研究。Lgt 的转酰基机制将为开发针对脂蛋白翻译后修饰途径中挑战性酶的新型抗菌疗法提供启示。
