TERPENE CYCLASES: FUNCTIONAL DOMAINS AND STRUCTURES

本研究的目的是了解四种植物倍半萜环化酶(包括烟草5-表马兜铃烯合成酶(TEAS)、杂色棉(Hyoscyamus Muticus VetiSpiRadiene Synthase)、亚洲棉(Gossypium Arboreum Cadinene Synthase)和青蒿L(Artemisia annua L epi-Cedrol Synthase))催化二磷酸法尼基环化反应的结构和机理。在之前的资助期间，野生型茶叶、突变茶酶和各种小分子络合物的X射线晶体结构提供了第一个植物萜类环化酶的原子分辨三维模型，使我们能够提出与FPP环化生成5-表马兜铃烯的化学合理性一致的酶反应机理。这些三维指南也是突变策略的起点，重点放在通过结晶学鉴定的有限活性部位残基上。最初的一组定点突变为我们提供了所提出的反应机制的确凿证据。我们现在准备扩展这一定向方法，将结构和生化信息与序列比对、同源建模和产物鉴定相结合，以了解萜类环酶底物和产物选择性的结构和机制基础。迅速膨胀的植物萜类环化酶序列数据库为我们提供了一些在环化酶活性位点上哪些位置变化最大的指示。我们建议使用单位置和多位置的定点突变、稳态和稳态前的动力学分析、银化薄层层析(Arg-TLC)和辐射气相色谱(r-GC)的产物分析、气相色谱/质谱仪(GC-MS)的产物鉴定和X射线结晶学来研究这种变异在底物选择和相关环化酶的替代反应机制中所起的作用。这一实验策略将作为在萜烯环酶中合理操作底物和产物专一性的起点。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化合物的努力。我们目前的目标是了解管理生物合成具有药用价值的萜类化合物的区域化学和立体化学原理。

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.