TERPENE CYCLASES: FUNCTIONAL DOMAINS AND STRUCTURES

本研究旨在了解烟草5-表-马兜铃烯合酶（TEAS）、天仙子香根草螺烯合酶（HVS）、亚洲棉杜松烯合酶（GCADS）和黄花蒿表-雪松醇合酶（AECS）等4种植物倍半萜环化酶催化法呢基二磷酸（FPP）环化反应的结构和机制特征。 在先前的资助期间，野生型TEAS、突变型TEAS酶和各种小分子复合物的X射线晶体结构提供了任何植物萜烯环化酶的第一个原子分辨率三维模型，这允许我们提出与FPP环化为5-表马兜铃烯的化学合理化一致的酶促反应机制。 这些三维指导物还用作诱变策略的起点，所述诱变策略集中于晶体学鉴定的有限的活性位点残基组。 最初的一组定点突变体为我们提供了坚实的证据所提出的反应机制。 我们现在定位于扩展这种定向方法，结合结构和生物化学信息与序列比对，同源性建模和产品鉴定，以了解萜烯环化酶的底物和产物选择性的结构和机制基础。 植物萜烯环化酶序列数据库的迅速扩大给了我们一些指示环化酶活性位点的位置是最可变的。 我们建议使用单点和多点定点突变，稳态和预稳态动力学分析，通过银化薄层色谱（arg-TLC）和放射性气相色谱（r-GC）进行产物分析，通过气相色谱/质谱（GC-MS）进行产物鉴定，和X-射线晶体学来说明这种可变性在底物选择和相关环化酶的替代反应机制中所起的作用。 这种实验策略将作为合理操作的底物和产品的萜烯环化酶的特异性的起点。 这些酶的底物和产物特异性的调节将直接影响生产具有治疗和农业意义的新型化合物的努力。 我们目前的目标是了解区域化学和立体化学的原则，管理生物合成的药用萜类化合物。

The objective of this proposal is to understand the structural and mechanistic features governing the cyclization of farnesyl diphosphate (FPP) catalyzed by a homologous set of four plant sesquiterpene cyclases including Nictotiana tabacum 5-epi-aristolochene synthase (TEAS), Hyoscyamus muticus vetispiradiene synthase (HVS), Gossypium arboreum cadinene synthase (GCADS), and Artemisia annua L epi-cedrol synthase (AECS). During the previous funding period, the x-ray crystal structures of wide-type TEAS, mutant TEAS enzymes, and various small molecule complexes with each provided the first atomic resolution three- dimensional models of any plant terpene cyclase that allowed us to propose an enzymatic reaction mechanism consistent with the chemical rationalization of FPP cyclization to 5-epi-aristolochene. These three- dimensional guides also served as starting points for a mutagenesis strategy focused on a limited set of active site residues identified crystallographically. The initial set of site-directed mutants have given us solid evidence for the proposed reaction mechanism. We are now positioned to expand this directed approach combining structural and biochemical information with sequence alignments, homology modeling, and product identification to understand the structural and mechanistic basis for both substrate and product selectivity in terpene cyclases. The rapidly expanding database of plant terpene cyclase sequences gives us some indiction of what positions in the cyclase active site are most variable. We propose to use site-directed mutagenesis at single and multiple positions, steady state and pre-steady state kinetic analysis, product profiling by argentation thin-layer chromatography (arg-TLC) and radiometric gas chromatography (r-GC), product identification by gas chromatography/mass spectrometry (GC-MS), and x-ray crystallography to address the role that this variability plays in substrate selection ad in the alternative reaction mechanisms of related cyclases. This experimental strategy will serve as starting points for the rational manipulation of the substrate and product specificity in terpene cyclases. Modulation of the substrate and product specificity of these enzymes will directly impact efforts to produce novel compounds of both therapeutic and agricultural interest. Our current objective is to understand the regiochemical and stereochemical principles that govern the biosynthesis of pharmaceutically useful terpenoids.