Limonene Hydroxylases as a Model for Cytochrome P450 Catalysis

萜类化合物是天然产物中最大的一类，在结构和生物功能上极其多样化。大多数萜类化合物是由一个共同的核心途径形成的，其中最初的环化反应决定了基本的结构类型，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(S)。氧化反应在转变为萜类化合物的最终产物中起主要作用，最具有相关生物学特性的是含氧的萜类化合物，而不是它们的碳氢化合物前体。P450硫化血红素酶是一大类催化剂，在生物合成和分解代谢过程以及解毒反应中都很重要。事实证明，这些酶的真核形式很难表征，活性部位结构的物理定义被排除在基于核磁共振的方法太大的范围之外，并且由于溶解的膜酶的顽固性而不能产生适合于X射线衍射的晶体。一个独特的信息模型系统，它利用了一组来自非常密切相关的薄荷物种的区域和立体特异性单萜羟基酶，已经在先前的NSF拨款下开发出来，这些重组酶为下一阶段的研究提供了基础，该研究涉及合成、生化、分子、建模和光谱工具，以解决关于这一重要酶类的活性部位结构-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基酶的功能性过表达系统；2.优化重组羟基酶的制备以用于光谱评估；3.使用空间定义的氟化和氚底物类似物以及电子-核双共振(Endor)光谱来确定不同羟基酶活性部位的底物结合几何构型；以及4.采用模型引导的突变，结合使用动力学探针，确定改变羟化区域和立体化学所需的活性部位结构的最小变化。这些实验目标的完成将检验这样一种假设，即活性部位地形的细微差异是生产形成的区域化学和立体化学变化的基础，并将提供其他类型的P450酶所不可能的结构理解的详细水平。此外，开发和重新设计这些重要的生物合成酶的能力将使它们能够用于困难的合成转化的绿色化学，并将提供一个前所未有的机会来设计植物中的萜类代谢途径。后者对操纵植物表现中的经济因素具有广泛的影响，从产生新产品，如药品、农用化学品和合成中间体，到抗病、抗虫害和抗除草剂。

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.