Life Sustaining Enzymes From Non-Natural Sequences

创造在体外具有催化活性和在体内具有生物学功能的新型酶是合成生物学的中心目标。该项目涵盖的研究通过设计和表征来自非天然氨基酸序列的生命维持酶来实现这一目标。实现这一目标很重要，原因有三：首先，这项研究的结果增强了对生命基本分子基础的理解。地球上的所有生命都来自共同的祖先，因此所有的生命系统都具有基本的相似性。这项研究考察了维持生命的酶，这些酶不是来自共同的祖先，而是从非天然氨基酸序列中设计、选择和进化而来的。这些新型蛋白质的表征揭示了来自非天然序列的酶是否只是简单地概括了自然界使用的结构和策略，或者（更令人兴奋的是）使用与共同祖先进化产生的序列、结构和机制不同的序列、结构和机制来维持生命。 其次，实现本项目的目标将扩大可用于实际应用的大分子的范围。第三，该项目的另一个目标是通过与人文主义者合作，向更广泛的人群解释进化论，以发展一种科学上准确的进化论，同时吸引精神上参与的人。该项目将通过以下步骤实现从非天然氨基酸序列设计和表征维持生命的酶的目标：（1）第一步是将潜在活性位点库设计到新蛋白质中。最近确定的第一个生命维持从头酶（Syn-F4）的晶体结构提供了一个支架的设计组合收集的侧链，探索数以百万计的替代催化位点的背景下，一个新的蛋白质结构。(2)然后对该文库进行遗传选择，以获得拯救E.杆菌(3)接下来，采用一系列遗传测定来阐明受这些新序列影响的生物学途径。这些分析的结果指向代谢途径和催化步骤。(4)最终，酶活性必须通过生物化学方法确认（和量化）。为了实现这一目标，从头酶的催化性能进行阐明纯化的蛋白质与底物及其类似物的动力学研究。(5)新蛋白质的三维结构得以解析，从而能够可视化新酶的活性位点。这些结构指导突变的构建，以确认新酶的拟议机制。(6)对新的（非进化的）序列进行基于实验室的进化以选择增强水平的酶活性。这些步骤的完成将导致一个新的酶，没有出现在自然界中，但仍然提供活动，维持生命。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

The creation of novel enzymes that are catalytically active in vitro and biologically functional in vivo is a central goal of synthetic biology. The research covered by this project achieves this goal by designing and characterizing life sustaining enzymes from non-natural amino acid sequences. Achieving this goal is important for three reasons: First, the results of this research enhance understanding of the fundamental molecular underpinnings of life. All life on Earth descended from common ancestry, and therefore all living systems share fundamental similarities. This research examines life-sustaining enzymes that did not descend from common ancestry, but instead are designed, selected, and evolved from non-natural amino acid sequences. Characterization of these novel proteins sheds light on whether enzymes from non-natural sequences simply recapitulate the structures and strategies used by nature, or alternatively (and more excitingly), sustain life using sequences, structures, and mechanisms that differ from those that arose by evolution from common ancestry. Second, achieving the goals of this project will broaden the range of macromolecules that can be used for practical applications. Third, an additional goal of the project focuses on explaining evolution to the broader population via collaborations with humanists to develop a discourse on evolution that is scientifically accurate, while simultaneously appealing to spiritually engaged people.The project will achieve the goal of designing and characterizing life-sustaining enzymes from non-natural amino acid sequences, by pursuing the following steps: (1) The first step is the design of a library of potential active sites into a novel protein. The recent determination of the crystal structure of the first life-sustaining de novo enzyme (Syn-F4) provides a scaffold for the design of a combinatorial collection of side chains that explore millions of alternative catalytic sites in the context of a novel protein structure. (2) This library is then subjected to genetic selections for life sustaining functions that rescue auxotrophic strains of E. coli. (3) Next, a series of genetic assays is employed to elucidate the biological pathway(s) impacted by these novel sequences. The results of these assays point towards metabolic pathways and catalytic steps. (4) Ultimately, enzymatic activity must be confirmed (and quantified) biochemically. Toward this goal, the catalytic properties of the de novo enzymes are elucidated by subjecting purified proteins to kinetic studies with substrates and their analogues. (5) three- dimensional structures of the new proteins are solved, thereby enabling visualization of the active sites of the novel enzymes. These structures guide the construction of mutations to confirm the proposed mechanisms of the novel enzymes. (6) The novel (non-evolved) sequences are subjected to laboratory- based evolution to select enhanced levels of enzymatic activity. Completion of these steps will lead to a collection of novel enzymes that did not arise in nature, but which nonetheless provide activities that sustain lifeThis award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.