Investigating neofunctionalized alpha-carbonic anhydrases as an emerging class of biosynthetic enzyme in plant and animal metabolism

摘要 自然代谢多样性是通过酶的新功能的进化而产生的 (新功能化)。社会利用这种新陈代谢的多样性来获得许多高价值的化学品，如 作为微生物和植物衍生的药物，但利用这种化学物质依赖于 潜在的生物合成机械。虽然一些生物合成酶很容易识别，但有 许多代谢反应没有明确的酶家族，这是澄清的障碍 新的代谢途径。我的实验室研究自然界的酶和化学反应， 侧重于鉴定药用植物中的生物合成基因和途径。我们特别是 有兴趣寻找新的酶，扩大已知代谢蛋白家族的‘目录’。 最近，我们发现了几种具有新功能的α-碳酸氢酶(Cah)样蛋白 在神经活性植物化合物的生物合成中催化新的支架形成反应。而当 这些是第一个被证明作为生物合成酶的CAH家族蛋白，我们预测 新功能化CAHs(neo-CAHs)在更广泛的代谢中具有关键的、未定义的功能。 在接下来的五年里，我的实验室将通过以下方式促进对新CAHs的基本理解 为它们的酶功能提供了机制基础，并通过研究它们的广度和多样性 新CAH酶在自然界中广泛存在。虽然经典的CAH已经得到了很好的研究，但生化 NEO-CAHs的性质尚未定义。我们将学习基础生物化学和催化 通过酶学特性、结构生物学和分析新的CAHs的机制 天然的翻译后修饰和亚细胞定位。这项工作将提供一种机械的 了解neo-CAH酶的催化作用，并将对用于 产生具有生物活性的植物分子。同时，我们将调查广泛发生的 自然界中的新CAHs。到目前为止发现的每个neo-CAH在保守的活性部位都有突变 残基对于规范的CAH功能是必不可少的。在其他基因中发现了类似的突变 植物、细菌和动物中未鉴定的CAH家族蛋白，表明CAH具有 不被欣赏的生物合成功能在多个生命王国中。我们将利用这些与众不同的优势 用于鉴定和功能表征其他neo-CAH酶的突变--包括来自 药用植物、微生物和人类--以更好地定义这个蛋白质家族的代谢能力。 通过这项工作，我们将a)提供对一类以前未知的代谢酶的洞察，该代谢酶 可能在自然界中具有更广泛的生物合成作用，以及b)进一步决定了保守的酶如何 获得新的功能，产生惊人的结构和功能多样性的天然代谢物。

ABSTRACT Natural metabolic diversity is generated through the evolution of novel function in enzymes (neofunctionalization). Society uses this metabolic diversity to obtain many high-value chemicals, such as microbial and plant-derived pharmaceuticals, but harnessing this chemistry relies on discovery of the underlying biosynthetic machinery. While some biosynthetic enzymes are readily identifiable, there are many metabolic reactions with no defined enzyme family, and this acts as a roadblock to elucidating new metabolic pathways. My lab studies enzymes and chemical reactions from the natural world, with a focus on identifying biosynthetic genes and pathways in medicinal plants. We are particularly interested in finding new enzymes that expand the ‘catalog’ of known metabolic protein families. Recently, we identified several α-carbonic anhydrase (CAH)-like proteins that have neofunctionalized to catalyze novel scaffold-forming reactions in the biosynthesis of neuroactive plant compounds. While these are the first CAH family proteins shown to act as biosynthetic enzymes, we predict that neofunctionalized CAHs (neo-CAHs) have critical, undefined functions in metabolism more broadly. Over the next five years, my lab will advance a fundamental understanding of neo-CAHs by providing a mechanistic basis on their enzymatic function and by investigating the breadth and diversity of neo-CAH enzymes throughout nature. While canonical CAHs are very well-studied, the biochemical properties of neo-CAHs are yet to be defined. We will study the foundational biochemistry and catalytic mechanisms of the neo-CAHs through enzymatic characterization, structural biology, and analysis of native post-translational modifications and sub-cellular localization. This work will provide a mechanistic understanding of neo-CAH enzyme catalysis and will yield basic insight into novel chemistry used to produce bioactive plant molecules. Simultaneously, we will investigate the widespread occurrence of neo-CAHs throughout nature. Each neo-CAH identified thus far has mutations in conserved active site residues that are essential for canonical CAH function. Similar mutations are found in other uncharacterized CAH family proteins within plants, bacteria, and animals, suggesting that CAHs have unappreciated biosynthetic functions in multiple kingdoms of life. We will leverage these distinguishing mutations to identify and functionally characterize other neo-CAH enzymes - including homologs from medicinal plants, microbes, and humans - to better define the metabolic capacity of this protein family. Through this work, we will a) provide insight on a previously unknown class of metabolic enzyme that likely has broader biosynthetic roles in nature, and b) further determine how conserved enzymes can gain new function to yield the striking structural and functional diversity of natural metabolites.