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，以促进低通量的途径，我们称之为“偶然途径”。当突变提高了通量水平，或者当环境发生变化，即使是低通量也能提高适应性或生存能力时，偶然途径就变得重要起来。这个提议解决了新的代谢途径的进化，这些途径是从混杂的酶活性中拼凑起来的。 这项工作对分子进化具有重要意义，无论是在过去，还是在现在，在应对人为化合物，如农药，弹药和化疗药物所施加的选择性压力。阿特拉津和五氯苯酚等农药的降解途径很可能来自于提供新的氮源或碳源或消除毒素的偶然途径。此外，在涉及非常大的群体（例如病原菌）或高突变率（例如肿瘤）的情况下，偶然的途径可能会促进对抗代谢药物的耐药性。 我们将探讨如何利用蛋白质组内的资源来组装偶然的途径，以及细胞如何适应在E.杆菌我们发现E.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酶缺失时，这些途径允许合成必需的辅因子磷酸吡哆醛。七种不同基因中的一种必须过度表达，以增加通过这些偶然途径的流量，达到支持生长的水平。我们描述了其中一条途径。目的1描述了表征第二种途径的努力，以进一步表征细胞内数千种混杂活性如何拼凑在一起以提供新的途径。 我们发现的偶然途径效率低下。此外，我们所描述的途径使用了两种不寻常的有毒代谢物。我们对18株大肠杆菌的基因组进行了重测序。大肠杆菌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描述了描述允许E.大肠杆菌更有效地利用偶然途径。 不同的微生物含有不同的酶。此外，不同微生物中的直系同源物之间的混杂活性水平可以显著变化。因此，将混杂活动结合到偶然途径中的潜力应该在微生物之间有所不同。目标3将研究如何利用肠道沙门氏菌代谢网络中的可用资源，以应对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.