STRUCTURE AND FUNCTION OF BIOSYNTHETIC ENZYMES

描述（由申请人提供）：自然界中发现的数千种萜烯和萜类衍生物参与了多种生物合成和代谢途径，如人类胆固醇的生物合成和太平洋红豆杉的紫杉醇（Taxol）合成。值得注意的是，由于其镇痛、抗生素和抗真菌的特性，许多萜类化合物自古以来就被用作药物。尽管这一天然产物家族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（又称合成酶）催化一种常见的烯丙基焦磷酸盐底物，如法尼酯二磷酸，形成数百种可能的产物之一的特定环化。萜类环化酶作为模板在“引导”精确的底物和中间构象从而形成一个专有产物方面起着关键作用。因此，从生物学和化学的角度来看，萜类环化酶构成了一类令人兴奋的生物合成酶。在目前的资助期内，我们已经确定了单萜环化酶(+)-龙脑二磷酸合酶的第一个x射线晶体结构；我们确定了一种倍半萜环化酶马兜铃烯合成酶的第5晶体结构；并且我们已经建立了由位点特异性的trichodiene合成酶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内，通过详细剖析trichodiene合成酶的结构-生物合成多样性关系，在这个杰出的基础上进一步发展。特定的!此外，我们将研究具有改变金属结合特性的位点特异性变体，我们还将研究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。为了扩大我们对萜类环化酶大家族的结构-功能关系的认识，我们还将研究二萜环化酶taxadiene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，这是治疗真菌感染的潜在药物靶点。现在我们已经在生成环萜烯产物的生物合成酶的研究中建立了坚实的基础，我们现在将研究一种利用环萜烯底物进行新型化学反应的酶，从而进一步丰富环萜烯天然产物的生物合成阵列。

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.