Section of Molecular Microbiology and Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom.
Department of Life Sciences, Imperial College London, London, United Kingdom.
J Biol Chem. 2021 Jan-Jun;296:100384. doi: 10.1016/j.jbc.2021.100384. Epub 2021 Feb 5.
UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition.
尿苷二磷酸葡萄糖-1-磷酸尿苷酰转移酶是一种将 UTP 和葡萄糖-1-磷酸转化为 UDP-葡萄糖的酶。在枯草芽孢杆菌 168 中，UDP-葡萄糖是用于将葡萄糖残基修饰到细胞壁磷壁酸（WTA）和形成葡脂的必需物质。在标准需氧生长条件下，B. 芽孢杆菌 UGPase GtaB 对于 UDP-葡萄糖的产生是必需的，而 gtaB 突变体表现出严重的生长和形态缺陷。然而，生物信息学预测表明，枯草芽孢杆菌中还存在另外两种 UTP-葡萄糖-1-磷酸尿苷酰转移酶。在这里，我们研究了其中一种名为 YngB 的功能。YngB 的晶体结构表明，该蛋白具有典型的折叠结构和所有必需的活性位点特征，是一种功能性 UGPase。此外，使用 UTP 和葡萄糖-1-磷酸作为底物，在体外可以证明 UGPase 活性。在 B. 芽孢杆菌 gtaB 突变体中，使用合成启动子表达 yngB 可导致 WTA 上重新引入葡萄糖残基并产生糖脂，表明该酶在体内可作为 UGPase 发挥作用。当 WT 和突变体枯草芽孢杆菌菌株在厌氧条件下生长时，可以检测到依赖于 YngB 的糖脂产生和 WTA 上的葡萄糖修饰，表明 YngB 在厌氧条件下从其天然启动子表达。基于这些发现，以及包含 yngB 的操纵子的结构和被认为是其表达所必需的转录因子，我们提出，除了 WTA 之外，其他细胞壁成分在氧气限制生长条件下可能也被葡萄糖残基修饰。
