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

铁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是由磷酸甘油或磷酸核糖醇的重复单位组成的。这种生物聚合物的普遍修饰是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）发挥作用。这种酯化过程部分中和阴离子聚合物，似乎对生物膜形成、细菌增殖和宿主细胞感染至关重要。d -丙烯酰载体蛋白连接酶DltA是一种类似于酰基辅酶a合成酶和萤火虫荧光素酶中发现的腺苷化结构域的酶。DltA催化atp驱动的d -丙氨酸的腺苷化，并将活化的d -丙烯酰转移到4'-磷酸antetheine的巯基上，该巯基共价连接在d -丙烯酰载体蛋白DltC的丝氨酸侧链上。整体膜蛋白DltB的作用尚未确定。DltD是一种具有n端跨膜锚点的膜结合蛋白，可能催化d -丙烯酰形式的DltC最终转移到磷壁酸。我们最近确定了革兰氏阳性细菌蜡样芽孢杆菌与d -丙氨酸腺苷酸和ATP复合物的DltA蛋白的三维结构。与ATP的配合物揭示了一种更为封闭的构象，这种构象以前在类似的腺苷化结构域中是未知的，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，以了解它们如何形成一个功能系统，并验证DltD是否作为d -丙烯酰转移酶催化钛酸d -丙烯酰化的最后一个反应步骤。从长远来看，从本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d -丙烯酰化途径变得合理，并有助于设计生物膜形成抑制剂。

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.