Most monoterpenes and sesquiterpenes are derived by cyclization of prenyl pyrophosphates to olefins followed by oxidative modification initiated by cytochrome P450-dependent hydroxylation. Studies with two sesquiterpene cyclases, differing in physical properties and reaction complexity, will test the hypothesis that the same active site residues play similar roles in the electrophilic mechanism of this enzyme type. Studies with three closely-related (-)-limonene hydroxylases, that differ in position of allylic hydroxylation of this simple monoterpene olefin, will test the proposal that minor alterations in active site topography underlie regiochemistry of oxygen insertion by these cytochrome P450 enzymes. Homogeneous proteins will be used to generate antibodies and partial amino acid sequences, and a molecular cloning and expression strategy adopted to obtain complete primary sequences and larger amounts of the enzymes. Radioactive modifying reagents will be employed to selectively label active site cysteine and histidine residues of the cyclases, and photolabile substrate analogs will be utilized to label hydrophobic regions of both cyclase and hydroxylase sites. Active site peptides will be prepared, sequenced, compared, and located on the proteins. The role of specific residues in cyclase catalysis will be determined by protection against chemical modification using analogs representing different substrate binding domains, and by directed mutagenesis. Limonene-tethered heme- directed inhibitors, as well as 19F-NMR methods, will be used to probe the spatial relationship between the heme iron of the cytochromes and the limonene substrate. These studies will provide new information on the nature of these novel cyclase and hydroxylase catalysts and a clearer understanding of the origin of terpenoid natural products. Terpenoids play an important role in the interaction of the plant with its environment. An understanding of terpenoid biochemistry will allow a deeper understanding of the role of these compounds in plants. In addition to the well known essential oils of resins of commerce, serious consideration is being given to nontraditional uses of these plant metabolites as renewable sources of energy- intensive chemicals. A broad range of terpenoids are of pharmacological significance and many are of agrochemical utility. New knowledge gained from this work will assist in efforts to increase the production of these valuable plant metabolites.//
