Elucidation and Evolutionary Potential of a Latent Pathway for PLP Synthesis

描述(由申请人提供)： 许多酶具有低效的混杂活性。混杂的活动可以拼凑在一起，以促进低流量的通路，我们称之为“偶然通路”。当突变提高通量水平时，或者当环境发生变化，即使是低通量也能改善健康或存活率时，偶然途径就变得重要起来。这项提议解决了新的代谢途径的进化，这些途径已经从混杂的酶活动中拼凑在一起。这项工作对分子进化具有重要意义，无论是在过去还是现在，都是为了应对杀虫剂、弹药和化疗药物等人为化合物施加的选择压力。阿特拉津和五氯苯酚等农药的降解途径可能来自偶然的途径，这些途径提供了一种新的氮源或碳源，或消除了一种毒素。此外，偶然途径可能会在涉及非常大的种群(例如致病菌)或高突变率(例如肿瘤)的情况下培养对抗代谢物药物的耐药性。我们将探索如何利用蛋白质组中的资源来组装偶然的途径，以及细胞如何适应在大肠杆菌模型系统的背景下更有效地使用这些途径。我们发现，大肠杆菌至少有三条偶然的途径，当正常生物合成途径中的一种酶缺失时，这三条途径允许合成必要的辅因子吡哆醛磷酸。七个不同基因中的一个必须过度表达，才能增加通过这些偶然途径的流量，达到支持生长的水平。我们已经描述了其中一条路径的特征。目标1描述了描述第二条途径的努力，以进一步描述细胞内数千种可用混杂活动如何拼接在一起以提供一条新的途径。我们发现的偶然路径是低效的。此外，我们所描述的途径使用了两种不寻常的有毒代谢物。我们已经对18株大肠杆菌的基因组进行了重新测序，这些菌株已经适应了在葡萄糖上更有效地生长，同时使用偶然的途径来供应PLP。目的2描述了描述突变的分子机制的计划，这些突变使大肠杆菌能够更有效地利用偶然途径。不同的微生物含有不同的酶补体。此外，不同微生物中的直系物之间的混杂活动水平可能会有很大差异。因此，将混杂活动结合到偶然途径中的可能性应该因微生物而异。目的3将研究如何利用肠沙门氏菌代谢网络中的可用资源来应对在正常途径被禁用时合成PLP的挑战。

DESCRIPTION (provided by applicant): Many enzymes have inefficient promiscuous activities. Promiscuous activities can be patched together to facilitate low-flux pathways that we have termed "serendipitous pathways". Serendipitous pathways become important when a mutation raises the level of flux or when the environment changes such that even low flux improves fitness or survival. This proposal addresses the evolution of new metabolic pathways that have been patched together from promiscuous enzyme activities. This work has important implications for molecular evolution, both in the past, and in the present in response to selective pressures exerted by anthropogenic compounds such as pesticides, munitions and chemotherapeutics. Pathways for degradation of pesticides such as atrazine and pentachlorophenol likely arose from serendipitous pathways that provided a novel source of nitrogen or carbon or eliminated a toxin. Further, serendipitous pathways might foster resistance to anti-metabolite drugs in situations involving very large populations (e.g. pathogenic bacteria) or high mutation rates (e.g. tumors). We will explore how the resources within a proteome can be utilized to assemble serendipitous pathways and how cells can adapt to use such pathways more efficiently in the context of a model system in E. coli. We have discovered that E. coli has at least three serendipitous pathways that allow synthesis of the essential cofactor pyridoxal phosphate when an enzyme in the normal biosynthetic pathway is missing. One of seven different genes must be over expressed to increase flux through these serendipitous pathways to a level that supports growth. We have characterized one of these pathways. Aim 1 describes efforts to characterize a second pathway to further characterize how the thousands of promiscuous activities available within a cell can be patched together to provide a novel pathway. The serendipitous pathways we have discovered are inefficient. Furthermore, the pathway we have characterized uses two unusual metabolites that are toxic. We have resequenced the genomes of 18 strains of E. coli that have been adapted to grow more efficiently on glucose while using serendipitous pathways to supply PLP. Aim 2 describes plans to characterize the molecular mechanisms of the mutations that allow E. coli to use a serendipitous pathway more effectively. Different microbes contain different complements of enzymes. Furthermore, the levels of promiscuous activities between orthologs in different microbes can vary significantly. Thus, the potential for combining promiscuous activities into serendipitous pathways should vary among microbes. Aim 3 will examine how the resources available in the metabolic network of Salmonella enterica can be used to meet the challenge of synthesizing PLP when the normal pathway is disabled.