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，并在动物环境毒素和植物除草剂的解毒中发挥重要作用。作为一类，细胞色素P450羟化酶一直很难研究，因为它们在X-射线结构测定中不容易结晶，并且它们在形式和反应类型上高度多样，从而提供了有限的机会来了解酶结构和特定功能之间的关系。薄荷属植物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一个独特的机会。三种不同的柠檬烯羟化酶已从常见的薄荷物种中克隆，其中每一种都在同一个非常简单的萜类底物柠檬烯上的不同位置上特异性地添加羟基。这些细胞色素P450酶在一级结构（氨基酸序列）上非常相似，并且很可能活性位点形状的非常细微的差异决定了底物上羟基化的具体位置。光敏底物类似物被用于定位活性位点，诱变研究被用于确定活性位点的哪些特定氨基酸指导羟基化的位置。柠檬烯的氟化类似物已经合成，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个天然酶和突变酶进行的反应相关联。 这些实验目标的完成将提供一个结构的理解，还没有可能与其他类型的细胞色素P450酶的水平。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，对生产新型植物产品（如药物，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的意义。 该项目涉及一类称为细胞色素P450的植物酶。它们催化植物产生重要的次生代谢产物如紫杉醇、防御介质和激素所必需的各种反应。此外，P-450还可以对污染物进行解毒，并分解除草剂等化合物。该项目将产生关于这些酶如何与其底物相互作用以指导不同产物合成的新信息。这项工作对代谢工程、生物修复和作物抗病性都有意义。

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.