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和萜类衍生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太平洋紫杉的紫杉醇合成。值得注意的是，许多萜类化合物自古以来就因其止痛、抗菌和抗真菌特性而被用作药剂。尽管这类天然产物对人类健康具有普遍的重要性，但值得注意的是，萜类环化酶的三维结构直到最近才被报道。萜类环化酶(又名合成酶)催化常见的烯丙基焦磷酸底物(如法尼基二磷酸)的特定环化反应，生成数百种可能的产物之一。萜类环化酶在引导精确底物和中间构象形成单一排他性产物的过程中起着关键的模板作用。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，我们已经确定了单萜环化酶(+)-龙脑二磷酸合成酶的第一个X射线晶体结构；我们已经确定了来自Terreus的倍半萜环化酶马兜铃烯合酶的第五个晶体结构；我们已经建立了毛状二烯合成酶的位点特异性变体形成异常产物的结构基础。我们的目标是在下一个资助期通过剖析毛状二烯合成酶的详细结构-生物合成多样性关系来建立这一杰出的基础。具体地说，我们将研究具有改变金属结合特性的特定位点变体，我们还将研究设计用于产生替代产品的变体。我们还将研究马兜铃烯合成酶的进化和保真度的结构基础。为了拓宽我们对萜类环化酶大家族结构与功能关系的认识，我们还将研究二萜环化酶紫杉二烯合成酶。最后，我们将确定甾醇甲基转移酶的X射线晶体结构，它是治疗真菌感染的潜在药物靶点。既然我们已经为产生环萜类产物的生物合成酶的研究奠定了坚实的基础，我们现在将研究一种利用环萜类底物的酶在一种新颖的化学反应中的应用，这将进一步使环萜类天然产物的生物合成阵列多样化。

DESCRIPTION (provided by applicant): Thousands of terpenes and terpenoid derivatives found throughout Nature are involved in diverse biosynthetic and metabolic pathways such as cholesterol biosynthesis in humans and paclitaxel (Taxol) synthesis in the Pacific yew. Notably, many terpenoids have been used as medicinal agents since 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, it is remarkable that the three-dimensional structures of terpenoid cyclases have only been reported relatively recently. Terpenoid cyclases (a.k.a. synthases) catalyze the specific cyclization of a common allylic pyrophosphate substrate, such as farnesyl diphosphate, into one of hundreds of possible products. The terpenoid cyclase plays a critical role as a template in "channeling" the precise substrate and intermediate conformations leading to the formation of one exclusive product. 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 first X-ray crystal structure of a monoterpene cyclase, (+)- bornyl diphosphate synthase; we have determined the fifth crystal structure of a sesquiterpene cyclase, aristolochene synthase from A.terreus; and we have established the structural basis for aberrant product formation by site-specific variants of trichodiene synthase. We aim to build upon this outstanding foundation in the next funding period by dissecting detailed structure-biosynthetic diversity relationships in trichodiene synthase. Specific!ally, we will study site-specific variants with altered metal binding properties, and we will also study variants engineered to generate alternative products. We will also study the structural basis for the evolution and fidelity of aristolochene synthase from A. terreus and P. roqueforti. In order to broaden our knowledge of structure-function relationships in the greater family of terpenoid cyclases, we will also study the diterpene cyclase taxadiene synthase. Finally, we will determine the X-ray crystal structure of sterol methyltransferase, a potential drug target for the treatment of fungal infections. Now that we have established a solid foundation in the study of biosynthetic enzymes that generate cyclic terpene products, we will now study an enzyme that utilizes a cyclic terpene substrate in a novel chemical reaction that further diversifies the biosynthetic array of cyclic terpene natural products.