Structure and Function of Biosynthetic Enzymes

描述(申请人提供)：自然界中发现的数以千计的萜类化合物参与多种生物合成和代谢途径，如人类的胆固醇生物合成和薄荷的薄荷醇生物合成。值得注意的是，许多萜类化合物自古以来就被用作药物，因为它们具有止痛、抗菌和抗真菌的特性。尽管这个天然产物家族对人类健康具有普遍的重要性，但萜类环化酶的三维结构直到最近才被报道；这些结构测定中的大多数都得到了GM56838的支持。萜类环化酶(又名萜烯合成酶)催化常见的烯丙基焦磷酸底物的环化反应，如法尼基二磷酸，形成数百种可能的产物之一。萜类环化酶在伴随底物和中间构象的过程中起着重要的模板作用。环化反应可以是非常特定的，并导致形成一个排他性产物，也可以是有点混杂的，导致几个产物的形成。因此，从生物和化学的角度来看，萜类环化酶构成了一类令人兴奋的生物合成酶。在目前的资助期间，我们已经确定了倍半萜环化酶马兜铃烯合酶、β-杜松烯合酶和表异齐扎烯合酶的X射线晶体结构；我们已经建立了野生型毛状二烯合酶及其定点突变体异常产物形成的结构基础；我们已经产生了二萜环化酶的第一个晶体--二磷酸铜合酶。我们的目标是在下一个资助期通过剖析马兜铃烯合成酶和表异氮烯合成酶的详细结构-功能关系来建立这一杰出的结构基础，以更好地了解生物合成多样性的结构基础。具体地说，我们将研究设计来产生替代产品的特定位点变体，并开发一种基于结构的方法来在蛋白质工程实验中产生新的环萜类化合物。此外，我们还将测定coalyl二磷酸合成酶和Geosmin合成酶的X射线晶体结构，以探索多结构域萜类环酶的结构演变。这些研究将阐明萜类合成酶大家族生物合成多样性的进化根源。 与公众健康相关：对萜类环化酶的结构和功能研究表明，这些新的酶如何产生地球上发现的最大和最多样化的天然产品家族。重要的是，许多萜类化合物显示出有用的药用特性，例如，作为抗菌、抗真菌、抗炎或抗癌药物。因此，了解和设计萜类环化酶在合成复杂碳支架中的功能具有很高的特异性和效率，最终将使合成化学和合成生物学之间的药物发现成为可能。

DESCRIPTION (provided by applicant): Thousands of terpenoid derivatives found throughout Nature are involved in diverse biosynthetic and metabolic pathways such as cholesterol biosynthesis in humans and menthol biosynthesis in mint. Notably, many terpenoids have been used as medicinal agents since the times of antiquity due to their analgesic, antibiotic, and antifungal properties. In spite of the universal importance of this family of natural products for human health, the three-dimensional structures of terpenoid cyclases have only been reported relatively recently; the majority of these structure determinations have been supported by GM56838. Terpenoid cyclases (a.k.a. terpene synthases) catalyze the cyclization of a common allylic pyrophosphate substrate, such as farnesyl diphosphate, to form one of hundreds of possible products. The terpenoid cyclase plays a critical role as a template in chaperoning substrate and intermediate conformations. The cyclization reaction can be very specific and lead to the formation of one exclusive product, or it can be somewhat promiscuous and lead to the formation of several products. Thus, the terpenoid cyclases comprise an exciting class of biosynthetic enzymes from both the biological and the chemical perspectives. In the current funding period, we have determined the X-ray crystal structures of the sesquiterpene cyclases A. terreus aristolochene synthase, delta-cadinene synthase, and epi-isozizaene synthase; we have established the structural basis for aberrant product formation by wild-type trichodiene synthase and its site-specific mutants; and we have generated the first crystals of a diterpene cyclase, copalyl diphosphate synthase. We aim to build upon this outstanding structural foundation in the next funding period by dissecting detailed structure-function relationships in aristolochene synthase and epi-isozizaene synthase to better understand the structural basis of biosynthetic diversity. Specifically, we will study site- specific variants engineered to generate alternative products, and we will develop a structure-based approach for generating new cyclic terpenoids in protein engineering experiments. Additionally, we will determine the X-ray crystal structures of copalyl diphosphate synthase and geosmin synthase to explore the evolution of domain architecture in multidomain terpenoid cyclases. These studies will illuminate the evolutionary roots of biosynthetic diversity in the greater family of terpenoid synthases. PUBLIC HEALTH RELEVANCE: Structural and functional studies of the terpenoid cyclases show how these novel enzymes generate the largest and most diverse family of natural products found on the Earth. Importantly, many terpenoids exhibit useful medicinal properties, e.g., as antibacterial, antifungal, anti-inflammatory, or anticancer agents. Therefore, understanding and engineering terpenoid cyclase function in generating complex carbon scaffolds with great specificity and efficiency will ultimately enable drug discovery at the interface of synthetic chemistry and synthetic biology.