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非天然氨基酸序列中的维持生命的酶来实现这一目标。实现这一目标很重要，原因有三：第一，这项研究的结果加强了对生命的基本分子基础的理解。地球上的所有生命都是共同祖先的后代，因此所有的生命系统都有基本的相似之处。这项研究考察了不是从共同祖先而来的维持生命的酶，而是从非天然氨基酸序列设计、选择和进化而来的。对这些新蛋白质的表征揭示了来自非自然序列的酶是简单地概括了大自然使用的结构和策略，还是使用不同于从共同祖先进化而来的序列、结构和机制来维持生命(更令人兴奋)。其次，实现这一项目的目标将拓宽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的合作，向更广泛的人群解释进化，以开发一种科学上准确的关于进化的论述，同时吸引精神上的参与者。该项目将通过以下步骤实现设计和表征非天然氨基酸序列中维持生命的酶的目标：(1)第一步是设计一个潜在活性位点的文库，将其转化为一种新的蛋白质。最近对第一个维持生命的从头酶(Syn-F4)的晶体结构的确定为设计一个侧链的组合集合提供了一个支架，这些侧链在一个新的蛋白质结构的背景下探索了数百万个替代的催化位点。(2)然后，该文库受到拯救营养缺陷性大肠杆菌菌株的维持生命功能的遗传选择的影响。(3)接下来，利用一系列的遗传分析来阐明受这些新序列影响的生物途径(S)。这些检测的结果指向代谢途径和催化步骤。(4)最终，酶的活性必须得到生化确认(和量化)。为了实现这一目标，通过对纯化的蛋白质与底物及其类似物进行动力学研究，阐明了从头酶的催化特性。(5)解决了新蛋白质的三维结构，从而能够可视化新酶的活性部位。这些结构指导突变的构建，以确认所提出的新型酶的机制。(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。完成这些步骤将产生一系列新的酶，这些酶不是自然界中产生的，但仍然提供维持生命的活动该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.