Pyridoxal 5'-phosphate homeostasis in Escherichia coli

后基因组时代的一个主要和未被充分认识的问题是， 数据库中一半蛋白质的分子功能是未知的。即使在大肠杆菌中 K12 MG 1655是研究最多的模式生物之一，约1200个基因仍是未知的（或 错误）功能。与此同时，代谢领域仍然存在悬而未决的问题，这些问题被认为是 像维生素的合成和回收一样得到很好的研究。例如，维生素B6以其活性形式作为 吡哆醛5 '-磷酸（PLP）是超过180种PLP依赖性酶的辅因子， 在许多代谢途径中。PLP是一种高度反应性的分子，既不稳定又有毒， 未与蛋白质结合的PLP的细胞内浓度保持较低。PLP如何交付给 在这些条件下的靶酶在所有生物体中仍然是一个谜。此外，虽然核心 E.大肠杆菌B6合成途径已被充分描述，参与补救的转运蛋白是 PLP合成/补救基因的调控尚未确定，且知之甚少。这个项目 重点是确定在大肠杆菌中PLP代谢中“缺失”的参与者。杆菌在这个过程中，函数 孤儿E.大肠杆菌基因应该得到解决，更普遍的是，关键保守的球员， PLP稳态特征。发表在两份手稿中的初步结果表明， 该E. coliYggS蛋白，COG 0325的成员以及其人直系同源物PROSC 与PLP依赖性疾病相关的疾病参与PLP体内平衡。的目标 目的1是检验这一假设，即这个家庭可能是在提供 PLP辅因子靶向酶。目标2的重点是通过识别 缺失E. coliB 6转运蛋白，并表征对高吡啶氧还蛋白水平的响应。最终 探索性的目标将集中在其他两个孤儿PLP结合蛋白和孤儿PLP结合蛋白。 调节器拟议的研究是重要的，因为在人类中，PLP或 PLP的缺乏或影响PLP代谢的缺陷与许多病理学有关，特别是 因此，了解PLP是如何传递到靶酶将具有 对人类疾病的广泛影响。此外，PLP代谢的许多酶具有 被认为是抗菌和抗寄生虫的目标，从而了解PLP的稳态 是开发成功抑制剂的关键。最后，这项工作应该表征5个基因， E.未知函数大肠杆菌，一种主要的模式生物。

One of the major and underappreciated issues of the post-genomic era is that the precise molecular functions of half of the proteins in databases are not known. Even in Escherichia coli K12 MG1655 one of the most studied model organism, ~1200 genes are still of unknown (or erroneous) function. In parallel, open questions remain in metabolic areas that were thought to be well studied like vitamin synthesis and salvage. For example, vitamin B6 in its active form as pyridoxal 5'-phosphate (PLP) is a cofactor for over 180 PLP-dependent enzymes that are involved in many metabolic pathways. PLP is a highly reactive molecule and is both labile and toxic so the intracellular concentration of PLP not bound to proteins remains low. How PLP is delivered to target enzymes in these conditions remains a mystery in all organisms. In addition, while the core E. coli B6 synthesis pathway is well described, the transporter(s) involved in salvage are unidentified and very little is known about regulation of PLP synthesis/salvage genes. This project focuses on identifying “missing” players in PLP metabolism in E. coli. In this process, the functions of several orphan E. coli genes should be solved and, more generally, key conserved players in PLP homeostasis characterized. Preliminary results published in two manuscripts have shown that the E. coli YggS protein, a member of the COG0325 as well as its human ortholog PROSC that has been linked to PLP-dependent diseases are involved in PLP homeostasis. The goal of Aim 1 is to test the hypothesis that this family could be the “missing” key player in delivering the PLP cofactor to target enzymes. Aim 2 focuses the response to PLP imbalance by identifying missing E. coli B6 transporters and characterizing the response to high Pyridoxin levels. The final exploratory aim will focus on two other orphan PLP binding proteins and an orphan PLP-binding regulator. The proposed research is significant because in humans, toxic levels of PLP or deficiency of PLP or defects affecting PLP metabolism are linked to many pathologies, particularly of the nervous system, hence understanding how PLP is delivered to target enzymes will have implications for a wide range of human diseases. Also, many enzymes of PLP metabolism have been recognized as antibacterial and antiparasitic targets, thus understanding PLP homeostasis is critical to develop successful inhibitors. Finally, this work should characterize 5 genes of unknown functions in E. coli, a major model organism.