TERPENE CYCLASES: FUNCTIONAL DOMAINS AND STRUCTURES

本研究的目的是了解四种同源植物倍半萜类环化酶，包括烟草5-马兜铃烯合成酶（TEAS）、木霉(Hyoscyamus muticus vetispiradiene合成酶（HVS）、木棉(Gossypium arboreum cadinene合成酶（GCADS）和黄花蒿(Artemisia annua L epi-cedrol合成酶（AECS），催化法尼酯二磷酸（FPP）环化的结构和机制特征。在之前的资助期内，宽型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，使我们能够提出与FPP环化到5-马铃子烯的化学合理化相一致的酶促反应机制。这些三维指南也可作为诱变策略的起点，聚焦于晶体学鉴定的有限活性位点残基集。最初的一组位点导向突变体为我们提出的反应机制提供了确凿的证据。我们现在的定位是扩展这种定向方法，将结构和生化信息与序列比对、同源性建模和产物鉴定相结合，以了解萜烯环化酶中底物和产物选择性的结构和机制基础。快速扩展的植物萜烯环化酶序列数据库为我们提供了环化酶活性位点中变化最大的位置的一些指示。我们建议使用单个和多个位点的定向突变，稳态和预稳态动力学分析，氩薄层色谱（arg-TLC）和辐射气相色谱（r-GC）的产物分析，气相色谱/质谱（GC-MS）的产物鉴定，以及x射线晶体学来解决这种变异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。该实验策略将作为合理操纵萜烯环化酶的底物和产物特异性的起点。这些酶的底物和产物特异性的调节将直接影响产生具有治疗和农业意义的新化合物的努力。我们目前的目标是了解区域化学和立体化学原理，这些原理支配着药用萜类化合物的生物合成。

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.