Structural and biochemical study on proteins involved in D-alanylation of teichoic acids

磷壁酸是在革兰氏阳性细菌中发现的一种独特的细胞壁成分。类似于核酸中的磷酸核糖骨架，这种阴离子生物聚合物由磷酸甘油或磷酸核糖醇的重复单元组成。这种生物聚合物的一个普遍的修饰是D-丙氨酸酯化，这需要由DLT基因编码的四种蛋白质(DltA、DltB、DltC和DltD)的功能。这一酯化过程部分中和了阴离子聚合物，对生物膜的形成、细菌增殖和宿主细胞感染似乎是必不可少的。D-丙氨酰载体蛋白连接酶DltA是一种类似于酰基辅酶A合成酶和萤火虫荧光素酶中的腺化结构域的酶。DltA催化由ATP驱动的D-丙氨酸的腺化，并将激活的D-丙氨基转移到4‘-磷酸丙氨酸的硫基上，该硫基共价连接到D-丙氨酰载体蛋白DltC的丝氨酸侧链上。完整的膜蛋白DltB的作用尚未确定。DltD是一种膜结合蛋白，其N-末端可能是跨膜锚点，它可能催化D-丙氨基从DltC到磷壁酸的最终转移。我们最近确定了一种来自革兰氏阳性杆菌蜡状芽孢杆菌的DltA蛋白与D-丙氨酸腺苷和三磷酸腺苷的复合体的三维结构。与ATP的络合物显示了以前未知的类似腺化结构域的更封闭的构象，这揭示了这些结构域的催化机制。我们建议进一步研究DltA的结构-功能关系，研究四种DLT蛋白之间可能的相互作用，以了解它们是如何形成一个功能系统的，并验证DltD是否作为催化硫壁酸D-丙氨酰化最后一步反应的D-丙氨酰转移酶。从长远来看，从这项研究中收集的信息将使这些蛋白质如何促进革兰氏阳性细菌中磷壁酸的这一重要的D-丙氨化途径合理化，并有助于生物膜形成抑制剂的设计。

Teichoic acid is a unique cell wall component found in Gram-positive bacteria. Similar to phosphoribose backbone in nucleic acids, this anionic biopolymer is made of repeating units of phosphoglycerol or phosphoribitol. A ubiquitous modification of this biopolymer is D-alanine esterification, which requires the functioning of four proteins (DltA, DltB, DltC and DltD) coded by the dlt gene. This esterification process partially neutralizes the anionic polymer, and appears to be essential for biofilm formation, bacterial proliferation and host cell infection. The D-alanyl carrier protein ligase DltA is an enzyme resembling the adenylation domains found in acyl-CoA synthetases and firefly luciferases. DltA catalyzes the ATP-driven adenylation of D-alanine and the transfer of the activated D-alanyl to the thiol group of 4'-phosphopantetheine which is covalently attached to a serine side chain of the D-alanyl carrier protein DltC. The role of the integral membrane protein DltB has not been established. DltD, a membrane-bound protein with an N-terminal putative transmembrane anchor, is a possible candidate for catalyzing the final transfer of D-alanyl form DltC to teichoic acid. We have recently determined the 3-dimensional structures of a DltA protein from a Gram-positive bacterium Bacillus cereus in complex with D-alanine adenylate and with ATP. The complex with ATP revealed a more closed conformation previously unknown to similar adenylation domains, which shed light on the catalytic mechanism of such domains. We proposed to further study the structure-function relationship of DltA, to study the possible interactions between the four Dlt proteins in order to understand how they form a functional system, and to verify whether DltD serves as the D-alanyl transferase which catalyzes the last reaction step of D-alanylation of teichoic acid. In the long run, information gleaned from this study would rationalize how these proteins promote this important D-alanylation pathway of teichoic acids in Gram-positive bacteria and aid the design of inhibitors of biofilms formation.