Pyridoxal 5'-phosphate homeostasis in Escherichia coli

后基因组时代的主要问题之一是，准确的 数据库中一半蛋白质的分子功能尚不清楚。即使在大肠杆菌中也是如此 K12 MG1655是研究最多的模式生物之一，~1200个基因仍然未知(或 错误)功能。同时，未解决的问题仍然存在于新陈代谢领域，这些领域被认为 在维生素合成和回收方面有很好的研究。例如，维生素B6的活性形式为 5‘-磷酸吡哆醛(PLP)是180多种依赖于PLP的酶的辅因子 在许多代谢途径中。PLP是一种高度活性的分子，既不稳定又有毒，所以 未与蛋白质结合的PLP的胞内浓度仍然很低。如何将PLP交付给 在这些条件下，靶标酶在所有生物体中仍然是一个谜。此外，虽然核心 E.ColiB6的合成途径已有描述，参与抢救的转运蛋白(S)有 对PLP合成/挽救基因的调控尚不清楚，也知之甚少。这个项目 重点识别在大肠杆菌中PLP新陈代谢中“缺失”的角色。在这个过程中，函数 几个孤儿大肠杆菌基因的问题应该解决，更广泛地说，关键的保守角色 PLP动态平衡的特征。在两篇手稿中发表的初步结果表明 COG0325的成员大肠杆菌YggS蛋白及其人类同源基因PROSC 这与PLP依赖疾病有关，参与了PLP的动态平衡。的目标是 目标1是检验这样一种假设，即这个家庭可能是传递 PLP辅因子作用于靶向酶。目标2重点关注PLP失衡的反应，通过确定 缺失E.ColiB6转运体并表征对高水平吡哆醇的反应。决赛 探索性目标将集中在另外两个孤儿PLP结合蛋白和一个孤儿PLP结合蛋白 调整器。这项拟议的研究意义重大，因为在人体内，PLP或PLP的毒性水平 PLP缺乏或影响PLP代谢的缺陷与许多病理有关，尤其是 因此，了解PLP是如何被输送到靶向酶的 对一系列人类疾病的影响。此外，PLP代谢的许多酶都有 被认为是抗菌和抗寄生虫的靶标，从而了解PLP的动态平衡 是开发成功的抑制剂的关键。最后，这项工作应该表征5个基因 主要的模式生物--大肠杆菌的未知功能。

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.