Limonene Hydroxylases as a Model for Cytochrome P450 Catalysis

萜类化合物是天然产物中最大的家族，其结构和生物学功能极其多样。大多数萜类化合物通过共同的核心途径形成，其中初始环化反应定义了基本结构类型，随后的细胞色素P450介导的羟基化反应决定了衍生代谢物家族的氧化模式。氧化反应在萜类终产物的多样化中起主要作用，并且氧化的萜类，而不是它们的烃前体，最经常具有相关的生物学性质。P450血红素硫醇化酶代表了在生物合成和分解代谢过程中以及在解毒反应中重要的非常大的一类催化剂。这些酶的真核形式已被证明难以表征，并且活性位点结构的物理定义已被排除，因为对于基于NMR的方法来说尺寸太大，并且溶解的膜酶的溶解性（absoluted membranous enzymes）不能产生适合于X射线衍射的晶体。一个独特的信息模型系统，利用了一套区域和立体特异性单萜羟化酶从非常密切相关的薄荷物种，已开发下一阶段的研究，涉及合成，生物化学，分子，建模和光谱工具，以解决有关这一重要酶类的活性位点结构-功能关系的基本问题。本项目的目标是：1.开发野生型和突变型羟化酶的功能性过表达系统; 2.优化重组羟化酶的制备，用于光谱评价; 3.使用空间限定的氟化和氘代底物类似物和电子-核双共振（ENDOR）光谱法确定不同羟化酶的活性位点处的底物结合几何结构;和4.采用模型引导的诱变，结合动力学探针的使用，以确定改变羟基化区域和立体化学所需的活性位点结构的最小变化。这些实验目标的完成将测试的假设，活性位点地形的细微差异的基础上的区域化学和立体化学的生产形成的改变，并将提供一个详细的结构水平的理解，还没有可能与其他类型的P450酶。此外，开发和重新设计这些重要的生物合成酶的能力将允许它们在“绿色”化学中用于困难的合成转化，并将提供前所未有的机会来工程化植物中的萜烯代谢途径。后者对操纵植物性能中的经济因素具有广泛的影响，范围从产生新产品，如药物，农用化学品和合成中间体，到疾病，昆虫和除草剂抗性。

Terpenoids constitute the largest family of natural products and are extremely diverse in structure and biological function. Most terpenoids are formed by a common core pathway in which an initial cyclization reaction defines the basic structural type and subsequent cytochrome P450-mediated hydroxylation reactions determine the oxygenation pattern(s) of the derived family of metabolites. The oxygenation reactions play primary roles in the diversification to terpenoid end products and it is the oxygenated terpenoids, rather than their hydrocarbon precursors, that most often possess the relevant biological properties. The P450 heme-thiolate enzymes represent a very large class of catalysts important in both biosynthetic and catabolic processes, as well as in detoxification reactions. Eukaryotic forms of these enzymes have proven difficult to characterize, and the physical definition of active site structure has been precluded by a size too large for NMR-based approaches and by the recalcitrance of the solubilized membranous enzymes to yield suitable crystals for X-ray diffraction. A uniquely informative model system, that exploits a set of regio and stereospecific monoterpene hydroxylases from very closely related mint species, has been developed under the prior NSF grant, and these recombinant enzymes provide the foundation for the next phase of the research which involves synthetic, biochemical, molecular, modeling and spectrometric tools to address fundamental questions concerning active site structure-function relationships of this important enzyme class. The objectives of this project are: 1. to develop a functional overexpression system for the wild-type and mutant hydroxylases; 2. to optimize the preparation of the recombinant hydroxylases for spectrometric evaluation; 3. to determine substrate binding geometry at the active sites of the different hydroxylases using spatially defined fluorinated and deuterated substrate analogs and electron-nuclear double resonance (ENDOR) spectrometry; and 4. to employ model-guided mutagenesis, coupled with the use of kinetic probes, to determine the minimum change in active site structure required to alter hydroxylation regio and stereochemistry. Completion of these experimental objectives will test the hypothesis that subtle differences in active site topography underlie alterations in the regiochemistry and stereochemistry of production formation, and will provide a detailed level of structural understanding that has not been possible with other types of P450 enzymes. Additionally, the ability to exploit and redesign these important biosynthetic enzymes will permit their use in 'green' chemistry for difficult synthetic transformations and will provide an unprecedented opportunity to engineer terpene metabolic pathways in plants. The latter has broad implications for the manipulation of economic factors in plant performance, ranging from the generation of novel products, such as pharmaceuticals, agrochemicals and synthetic intermediates, to disease, insect and herbicide resistance.