ENZYMATIC ACTIVATION OF LIPOPHILIC SIGNALING MOLECULES

Louisiana State Univ A&M Col Baton Rouge, Baton Rouge LA

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Abstract

Description: (Applicant's abstract) Retinoids and eicosanoids are mediators of numerous biological processes and as such are compounds of enormous pharmacological potential. Structural studies on proteins which catalyze the activation of these compounds, which readily partition into the cell membrane, to their potent biological forms will add to our understanding of substrate recognition and acquisition, two themes directly addressed by experiments in this proposal. Two contrasting mechanisms of substrate acquisition are represented herein: one which utilizes a freely soluble carrier protein for delivery of the substrate to an activating enzyme (intermolecular) and one in which two sequential catalytic activities are expressed on a single polypeptide and consequently co-localized (intramolecular). The following proposed aims involve x-ray crystallographic studies on three enzymes: retinol dehydrogenase, retinol dehydratase, and allene oxide synthase. The specific questions to be addressed are: (1) Does substrate binding order the substrate access channel and catalytic machinery of retinaldehyde dehydrogenase? Both crystallographic and kinetic experiments are proposed to elucidate the mechanism of substrate recognition by this enzyme. (2) Is protein:protein recognition involved in substrate recognition by retinal dehydrogenase? In vivo retinol and retina1 are protein bound. Both crystallographic and solution studies are proposed to determine how RalDH2 interacts with the carrier protein for retinol. (3) Determination of the structure and mechanism of retinol dehydratase, a sulfotransferase which catalyzes the dehydration of retinol. (4) Determination of the three dimensional structural of the allene oxide synthase domain, a unique member of a catalase super-family of heme enzymes. A naturally occurring fusion protein which contains a lipoxygenase domain and an allene oxide synthase domain has been identified. The lipoxygenase domain catalyzes the production of 8-hydroperoxyeicsoatetraenoic acid (8-HPETE) from arachidonic acid and the allene oxide synthase domain catalyzes the transformation of the hydroperoxide to an allene oxide. The expression of this activity as a fusion protein with lipoxygenase is predicted to facilitate the transfer of the reaction intermediate between catalytic sites.