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.
