Evolutionary Insights Into Enzyme Mechanisms

描述（由申请人提供）：酶催化生物学必需的反应。从广义上讲，研究人员知道它们是如何工作的，但不知道它们是如何工作的。酶的复杂性使得很难确定哪些部分对催化作用很重要。反应位点的氨基酸是必需的，但不足以产生高活性的酶。远距离残基也很重要，一些残基仅在某些情况下有帮助，当其他残基也存在时（上位性）。确定所有有助于催化的残基是了解酶为什么工作良好的重要第一步。为了确定这些残基，我们建议重新创建祖先酶。这种重建降低了复杂性，因为它集中在那些与历史功能变化直接相关的少数突变上，从而避免了陷阱 使用（不完整的）基于知识的方法所固有的。需要筛选的差异数量从数百个减少到数十个，使得能够系统地使用定点诱变来探索每个突变对功能变化的贡献。祖先序列重建还具有鉴定上位性位点的潜力， 形成局部最优，定向进化方法陷入其中。超家族中功能的平行获取和逆转允许探索替代解决方案。初步数据表明，这种方法是可行的：祖先酶已被合成，表现出活性和更混杂比现代专业的后代和网站的关键以外的高活性sites已经found.Recreating催化混杂的祖先“干细胞”酶使我们能够夺回功能可塑性需要确定什么决定不同类型的活动在同一个蛋白质折叠。 公共卫生相关性：酶催化生命所必需的反应，当它们不能正常工作时，疾病是不可避免的，例如人类新陈代谢的先天性缺陷。阐明酶的作用机制为合理的基于机制的药物设计和重新设计酶以用于非自然应用开辟了道路，包括药物合成和“绿色”化学，其避免了昂贵的污染控制和生物修复的需要。我们的研究重点是为制药行业和人类健康开发重新设计酶的方法。

DESCRIPTION (provided by applicant): Enzymes catalyze the reactions essential to biology. In broad strokes, researchers understand how they work, but not how they work well. The complexity of enzymes makes it hard to identify which parts are important to catalysis. The amino acids in the reaction site are essential, but not enough to create highly active enzymes. Distant residues are also important and some residues are helpful only in some cases, when other residues are also present (epistasis). Identifying all residues that contribute to catalysis s an essential first step to learn why enzymes work well. To identify these residues, we propose to recreate ancestral enzymes. This recreation reduces complexity because it focuses on those few mutations directly associated with historical functional changes, thereby avoiding the pitfalls inherent to using (incomplete) knowledge based approaches. The number of differences that need to be screened is reduced from hundreds to tens enabling the systematic use of site directed mutagenesis to explore the contribution that each mutation makes to changes in function. Ancestral sequence reconstruction also has the potential of identify epistatic sites that form local optima on which directed evolution methods get trapped. Parallel acquisitions and reversals in function within superfamilies allow alternative solutions to be explored. Preliminary data show that the approach is feasible: ancestral enzymes have been synthesized, shown to be active and more promiscuous than modern specialist descendants and sites critical to high activity outside active sites have been found. Recreating catalytically promiscuous ancestral "stem cell" enzymes allows us to recapture the functional plasticity needed to identify what determines different types of activity in the same protein fold. PUBLIC HEALTH RELEVANCE: Enzymes catalyze the reactions essential to life and when they do not work properly disease is inevitable, e.g. inborn errors of human metabolism. Elucidating mechanisms of enzyme action opens the way to rational mechanism-based drug design and redesigning enzymes for unnatural applications, including drug synthesis and 'green' chemistries that obviate the need for expensive pollution controls and bioremediation. Our research focuses on developing methodologies for redesigning enzymes for the pharmaceutical industry and for human health.