STRUCTURE AND FUNCTION OF BIOSYNTHETIC ENZYMES

描述（由申请人提供）：在自然界中发现的数千种萜烯和萜类衍生物参与多种生物合成和代谢途径，如人类胆固醇生物合成和太平洋紫杉醇（Taxol）合成。值得注意的是，许多萜类化合物由于其镇痛、抗生素和抗真菌特性，自古以来就被用作药剂。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，萜类化合物环化酶的三维结构只是最近才报道的。萜类环化酶（也称为酶）催化常见的烯丙基焦磷酸底物（如法呢基二磷酸）的特异性环化为数百种可能产物中的一种。萜类化合物环化酶作为模板在“引导”精确底物和中间构象中起着关键作用，导致一种独特产物的形成。因此，萜类化合物环化酶从生物学和化学的角度都包括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的第一个X-射线晶体结构，（+）-藜芦基二磷酸合酶;我们已经确定了倍半萜环化酶的第五个晶体结构，A.terreus的马兜铃烯合酶;我们已经建立了由马兜铃二烯合酶的位点特异性变体形成异常产物的结构基础。我们的目标是建立在这个优秀的基础上，在下一个资助期解剖详细的结构，生物合成的多样性关系，在双烯合酶。具体点！此外，我们将研究具有改变的金属结合特性的位点特异性变体，我们还将研究经工程改造以产生替代产品的变体。我们还将研究马兜铃烯合酶的进化和保真度的结构基础。terreus和P. roqueforti。为了拓宽我们对萜类环化酶大家族结构-功能关系的认识，我们还将研究二萜环化酶紫杉二烯合酶。最后，我们将确定甾醇甲基转移酶的X-射线晶体结构，这是一种治疗真菌感染的潜在药物靶点。既然我们已经在产生环状萜烯产物的生物合成酶的研究中建立了坚实的基础，我们现在将研究在新的化学反应中利用环状萜烯底物的酶，该化学反应进一步使环状萜烯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.