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The general area of interest in our laboratory is the biochemistry and molecular biology of higher plants. Specific areas currently under study are the mechanism of ethylene biosynthesis, an important phytohormone and cytochrome P450 monooxygenase mediated secondary metabolism in plants. We have cloned and overexpressed in E. coli 1-aminocyclopropane-1-carboxylic acid oxidase (ethylene forming enzyme) from avocado. This Fe (II) and ascorbate requiring oxidase is responsible for the last step of ethylene biosynthesis in which 1-aminocyclopropane-1-carboxylic acid (ACC) is converted to ethylene. We are currently investigating the kinetic mechanism of ACC oxidase; exploring the active site using fluorescence quenching of endogenous tryptophans; and examining various ligand-bound states of enzyme:Fe(II):NO complexes in using electrons paramagnetic resonance (EPR). These studies are being done in collaboration with Dr. N. O. Reich of the Chemistry Department. We have characterized a number of cDNA clones that represent ripening-related mRNAs from avocado fruit. One of these encodes a cytochrome P450 monooxygenase designated CYP71A1. The general role of P450 enzymes is to hydroxylate hydrophobic compounds, although the specific role of the CYP71A1 gene in fruit ripening is unknown and currently under investigation. The heterologous expression of the CYP71A1 protein in yeast has allowed in vitro characterization of the CYP71A1 protein. We have shown that various monoterpene compounds are either hydroxylated or epoxydated by the CYP71A1 enzyme. This suggests that the endogenous substrate may be related to monoterpenes, a class of compounds known to be important to fruit aroma or flavor. We have also started to explore the involvement of P450 genes in the metabolic functions of Arabidopsis thaliana, a plant system much more amenable to molecular genetic manipulations than avocado. |
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