Metabolic pathways for production and recycling of the visual chromophore, retinal, are essential for vision. In vertebrates, the key recycling reaction, namely trans-to-cis isomerization, depends on the retinal pigmented epithelium protein, RPE65. Mutations in RPE65 lead to a spectrum of retinal dystrophies ranging from Leber congenital amaurosis (LCA) to autosomal recessive retinitis pigmentosa (RP). Aside from null mutations, RPE65 missense mutations may affect protein stability, catalytic activity and membrane association. However, without sufficient structural information it is impossible to truly understand the functioning of RPE65 and the consequences of these mutations. Recent biochemical evidence indicates that the RPE65-related insect carotenoid-oxygenase, NinaB, catalyzes a combined carotenoid cleavage and trans-to-cis isomerization reaction. Thus, comparing the structures of RPE65 and NinaB constitutes a unique challenge that will contribute to our understanding of RPE65 function and help delineate the consequences of amino acid substitutions in this critical enzyme. The robust enzymatic activity of NinaB will allow translating structural predications into functional testing of key residues for trans-to-cis-isomerization, oxidative cleavage, enzyme/substrate and membrane interactions for this class of proteins. In this context we propose three specific aims involving structural, biochemical and physiological approaches to analyze the structure and function of retinoid- and carotenoid-converting enzymes.
In Aim 1, we will compare the molecular structures of RPE65 and NinaB. Determining the crystal structure of native RPE65 at 2.14 E resolution was a breakthrough critical to this endeavor. Now we propose to determine the structure of recombinant NinaB. Comparing structures of RPE65 and NinaB will allow identification of critical residues related to the oxidative cleavage reaction, isomerase reaction, substrate interactions and membrane association.
In Aim 2, the structural basis for membrane association of RPE65 and NinaB will be elucidated. Based on the structural analysis of RPE65, we will test the roles of palmitoylation, hydrophobic protein/membrane interactions and dimerization.
In Aim 3, a structure-based dissection of isomerase and oxygenase activities will be accomplished. Due to structural conservation of carotenoid-oxygenases and RPE65, comparisons of the substrate binding regions provide an opportunity to indentify amino acid residues required for the all-trans to 11-cis isomerase reaction. Aside from contributing fundamentally to understanding the chemistry of vision, this approach will help delineate the consequences of amino acid substitutions in these enzymes that are associated with impaired ocular vitamin A metabolism. Such knowledge also could translate into improved therapies for patients with disabling mutations in RPE65.
Comprehensive knowledge of biological structures and molecular events that comprise their mechanism of action is needed to improve our understanding and treatment of retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). But molecular understanding of key enzymes in the visual cycle is still lacking. The objective of this research is to elucidate the pathway for chromophore production from carotenoids including its trans-to-cis isomerization.
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