Recruitment of Enzymes to Serve New Functions

描述(由申请人提供)： 在地球生命近40亿年的历史中，招募蛋白质来发挥新的功能在代谢多样性的演变中发挥了关键作用。随着细菌对人类活动施加的新的选择压力做出反应，这一过程仍然很重要。对人类健康特别重要的是抗生素耐药性的出现，这种耐药性可能是通过招募先前存在的蛋白质来修改新引入的抗生素而产生的。此外，招募蛋白质在有毒的人为化合物的生物降解中发挥新的功能对于从环境中去除这些毒素至关重要。 虽然很明显已经发生了招募蛋白质来服务于新功能的情况，但对这一过程的研究很少。这项建议描述了对酶的招募过程的研究，以服务于对大肠杆菌的生存至关重要的各种功能。这项工作的目标是鉴定大肠杆菌基因组中的基因，对于这些基因，过度表达或突变可以通过编码可被招募来服务于缺失酶功能的蛋白质来恢复缺乏关键代谢酶的突变株的生存能力。以下类别的酶将成为目标：1)催化非常简单的反应，如脱水、醇的氧化和磷酰基转移的酶；2)具有共同结构折叠的酶，对于这些酶，可能会招募许多具有类似折叠的酶来取代缺失的酶；以及3)具有不常见的结构折叠的酶，对于这种酶，可能需要细菌部分更多的创造力来寻找替代品。进一步的研究将确定招募的蛋白质中靶向酶活性的水平，以及它的原始功能和结构折叠。对于选定的案例，将使用体外进化来尝试提高招募蛋白质中的靶向酶活性水平。 这项工作的结果将是对招募过程和现有蛋白质支架中可用催化可塑性的扩展视图。这项工作将深入了解大自然如何利用这种可塑性来进化新的代谢能力，并将提供有关某些蛋白质支架的催化能力的新信息，蛋白质工程师可以利用这些信息来创造新型催化剂。

DESCRIPTION (provided by applicant): Recruitment of proteins to serve new functions has played a key role in the evolution of metabolic diversity during the nearly 4 billion-year history of life on Earth. This process continues to be important as bacteria respond to novel selection pressures imposed by human activities. Of particular importance to human health is the emergence of antibiotic resistance, which can arise by recruitment of a pre-existing protein to modify a newly introduced antibiotic. In addition, the recruitment of proteins to serve new functions in the biodegradation of toxic anthropogenic compounds is critical for removal of these toxins from the environment. Although it is clear that recruitment of proteins to serve new functions has occurred, the process has rarely been studied. This proposal describes studies of the process of recruitment of enzymes to serve a variety of functions that are critical to the survival of E. coli. The goal of this work is identification of genes in the E. coli genome for which over expression or mutation restores viability in mutant strains lacking critical metabolic enzymes by encoding proteins that can be recruited to serve the function of the missing enzyme. The following classes of enzymes will be targeted: 1) enzymes that catalyze very simple reactions, such as dehydration, oxidation of an alcohol, and phosphoryl transfer; 2) enzymes that have a common structural fold and for which many enzymes with a similar fold might be recruited to replace the missing enzyme; and 3) enzymes that have an uncommon structural fold, for which more creativity on the part of the bacterium may be required to find a replacement. Further studies will determine the level of the targeted enzyme activity in the recruited protein, as well as its original function and structural fold. For selected cases, in vitro evolution will be used to attempt to improve the level of the targeted enzyme activity in the recruited protein. The outcome of this work will be an expanded view of the process of recruitment and of the catalytic plasticity available in existing protein scaffolds. This work will provide insights into how Nature has exploited this plasticity to evolve new metabolic capabilities and will provide new information about the catalytic capabilities of certain protein scaffolds that can be exploited by protein engineers to create novel catalysts.