Limonene Hydroxylases as a Model for Cytochrome P450 Catalysis

细胞色素P450羟化酶是植物和动物中一类重要的酶。它们负责植物和动物激素形成的关键生物合成步骤，并在动物的环境毒素解毒和植物的除草剂中发挥重要作用。细胞色素P450羟化酶是一类难以研究的酶，因为它们难以结晶以进行x射线结构测定，并且它们的形式和反应类型高度多样化，因此了解酶结构与特定功能之间关系的机会有限。薄荷物种的柠檬烯羟化酶为探索酶的结构特征如何决定催化反应的三维过程提供了独特的机会。从常见的薄荷中克隆出三种不同的柠檬烯羟化酶，每一种都在相同的、非常简单的萜类底物柠檬烯的不同位置上特异性地添加一个羟基。这些细胞色素P450酶在一级结构（氨基酸序列）上非常相似，很可能活性位点形状的细微差异决定了羟基化在底物上的特定位置。光活性底物类似物被用来定位活性位点，诱变研究被用来确定活性位点的哪些特定氨基酸指导羟基化的位置。已经合成了柠檬烯的氟化类似物，并将与核磁共振光谱法结合使用，以将底物的精确结合几何形状与每种天然酶和突变酶进行的反应联系起来。这些实验目标的完成将提供其他类型的细胞色素P450酶无法实现的结构理解水平。所获得的信息可能允许操纵和重新设计植物中的重要代谢途径，对生产新的植物产品，如药品、农用化学品和工业中间体，以及改善作物物种的疾病、昆虫和除草剂抗性具有广泛意义。该项目涉及一类称为细胞色素p450的植物酶。它们催化各种反应，这些反应对植物产生重要的次生代谢物如紫杉醇、防御介质和激素是必要的。此外，p -450可以解毒污染物和分解化合物，如除草剂。这个项目将产生新的信息，关于这些酶如何与底物相互作用，以指导不同产品的合成。这项工作对代谢工程、生物修复和作物抗病具有指导意义。

9604918 Croteau The cytochrome P450 hydroxylases are an important class of enzymes in both plants and animals. They are responsible for key biosynthetic steps in the formation of plant and animal hormones and play essential roles in detoxification of environmental toxins in animals and herbicides in plants. As a class, the cytochrome P450 hydroxylases have been difficult to study because they are recalcitrant to crystallization for x-ray structure determination and they are highly diverse in form and in reaction type, thus providing limited opportunity to understand the relationship between enzyme structure and specific function. The limonene hydroxylases of mint species provide a unique opportunity for exploring how enzyme structural features determine the three-dimensional course of the reaction catalyzed. Three distinct limonene hydroxylases have been cloned from common mint species, each of which carries out the specific addition of a hydroxyl group to a different position on the same, very simple terpenoid substrate limonene. These cytochrome P450 enzymes are very similar in primary structure (amino acid sequence), and it is likely that very subtle differences in the shape of the active sites determine the specific position of hydroxylation on the substrate. Photoactive substrate analogs are being used to locate the active sites, and mutagenesis studies are being employed to determine which specific amino acids of the active site direct the position of hydroxylation. Fluorinated analogs of limonene have been synthesized and will be used in conjunction with nuclear magnetic resonance spectrometry to correlate the precise binding geometry of the substrate with the reaction carried out by each native and mutant enzyme. Completion of these experimental objectives will provide a level of structural understanding that has not been possible with other types of cytochrome P450 enzymes. The information gained may allow the manipulation and redesign of important metabolic pathways in plants, broad implications for the production of novel plant products, such as pharmaceuticals, agrochemicals and industrial intermediates, and for the improvement of disease, insect, and herbicide resistance of crop species. This project is concerned with a class of plant enzymes called cytochrome P450s. They catalyze a variety of reactions that are necessary for plants to make important secondary metabolites such as taxol, defense mediators, and hormones. In addition, P-450s can detoxify pollutants and breakdown compounds such as herbicides. This project will result in new information about exactly how these enzymes interact with their substrates to direct synthesis of diverse products. The work has implications for metabolic engineering, bioremediation, and crop disease resistance.