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

磷壁酸是在革兰氏阳性菌中发现的独特细胞壁组分。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。 这种生物聚合物的普遍修饰是D-丙氨酸酯化，其需要由dlt基因编码的四种蛋白质（DltA、DltB、DltC和DltD）的功能。该酯化过程部分中和阴离子聚合物，并且似乎是生物膜形成、细菌增殖和宿主细胞感染所必需的。D-丙氨酰载体蛋白连接酶DltA是一种类似于酰基辅酶A合成酶和萤火虫脱氢酶中发现的腺苷酸化结构域的酶。 DltA催化D-丙氨酸的ATP驱动的腺苷酸化和活化的D-丙氨酰转移至4 ′-磷酸泛酰巯基乙胺的巯基，所述巯基共价连接至D-丙氨酰载体蛋白DltC的丝氨酸侧链。膜整合蛋白DltB的作用尚未确定。DltD是一种具有N-末端跨膜锚的膜结合蛋白，是催化D-丙氨酰形式DltC最终转移为磷壁酸的可能候选物。我们最近已经确定了从革兰氏阳性细菌蜡状芽孢杆菌与D-丙氨酸腺苷酸和ATP的复合物的DltA蛋白的三维结构。与ATP的复合物揭示了一个更封闭的构象以前未知的类似的腺苷酸化结构域，这揭示了这种结构域的催化机制。我们建议进一步研究DltA的结构-功能关系，研究Dlt蛋白之间可能的相互作用，以了解它们如何形成一个功能系统，并验证DltD是否作为D-丙氨酰转移酶催化磷壁酸的D-丙氨酰化的最后一个反应步骤。从长远来看，从这项研究中收集的信息将合理化这些蛋白质如何促进革兰氏阳性细菌中磷壁酸的这种重要的D-丙氨酰化途径，并有助于设计生物膜形成的抑制剂。

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.