Carotenoid cleavage dioxygenases (CCDs) are a unique family of non-heme iron oxygenases. The human genome encodes three family members, namely BCO1, BCO2, and RPE65 that are critical for carotenoid and retinoid metabolism related to vision. Thus, mutations in their genes are associated with blindness, vitamin A deficiency and altered carotenoid plasma levels. Emerging evidence indicates that these enzymes are targets for retinoid cycle modulators, a drug class that can correct ocular disease states related to retinoid metabolism. Here we present studies designed to unveil the biochemical and structural basis of the substrate specificity and catalytic mechanism of these important enzymes. Dissecting molecular mechanisms underlying CCDs' functions will advance our knowledge of biochemical processes that govern this disease-associated metabolism and will assist the design of therapeutics directed against ocular disease states.
In Aim 1, we will determine the architecture of the enzyme/substrate complex at an atomic scale. Properly folded but enzymatically inactive metal-substituted CCDs will be crystallized in the presence and absence of bound substrate/inhibitors. Spectroscopy techniques will be used to survey the metalloprotein substrate arrangement in the active center. These analyses will provide a wealth of information about the oxygen activation process and changes in metal oxidation state during the reaction. Together with 18O labeling experiments, these studies will provide unprecedented insights into the catalytic mechanism of these enzymes.
In Aim 2, we will unveil the biochemical and structural basis of vitamin A production. In this pathway, BCO1 and BCO2 must distinguish between more than 600 structurally related naturally occurring carotenoids. We will employ recombinant purified enzymes and natural and synthetic substrates to determine the selectivity of these enzymes for a specific ionone ring site of these bicyclic substrates. Information about the architecture of the protein-substrate complex will allow us to determine critical amino acid residues that account for the stereo- and region-specificity of CCDs. With the help of unique knockout mouse models we will translate this knowledge into the in vivo situation and study how deficiency of either CCD can affect ocular retinoid metabolism and functioning.

Public Health Relevance

Increasing evidence indicates that the protein family of carotenoid cleavage dioxygenases plays a pivotal role in carotenoid and vitamin A metabolism related to vision. These enzymes constitute also potential targets for drugs that correct common retinal degenerative diseases. Thus, dissecting molecular mechanisms underlying the enzymes' functions will pave the way for more effective treatments of human retinal degenerative diseases.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY020551-05A1
Application #
9104990
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Neuhold, Lisa
Project Start
2009-11-01
Project End
2020-03-31
Budget Start
2016-04-01
Budget End
2017-03-31
Support Year
5
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Widjaja-Adhi, Made Airanthi K; Ramkumar, Srinivasagan; von Lintig, Johannes (2018) Protective role of carotenoids in the visual cycle. FASEB J :fj201800467R
Sui, Xuewu; Farquhar, Erik R; Hill, Hannah E et al. (2018) Preparation and characterization of metal-substituted carotenoid cleavage oxygenases. J Biol Inorg Chem 23:887-901
Arreguín, A; Ribot, J; Mušinovi?, H et al. (2018) Dietary vitamin A impacts DNA methylation patterns of adipogenesis-related genes in suckling rats. Arch Biochem Biophys 650:75-84
Kelly, Mary E; Ramkumar, Srinivasagan; Sun, Weizhong et al. (2018) The Biochemical Basis of Vitamin A Production from the Asymmetric Carotenoid ?-Cryptoxanthin. ACS Chem Biol 13:2121-2129
Gao, Songqi; Kahremany, Shirin; Zhang, Jianye et al. (2018) Retinal-chitosan Conjugates Effectively Deliver Active Chromophores to Retinal Photoreceptor Cells in Blind Mice and Dogs. Mol Pharmacol 93:438-452
Xu, Mingchu; Xie, Yajing Angela; Abouzeid, Hana et al. (2017) Mutations in the Spliceosome Component CWC27 Cause Retinal Degeneration with or without Additional Developmental Anomalies. Am J Hum Genet 100:592-604
Sun, Da; Sahu, Bhubanananda; Gao, Songqi et al. (2017) Targeted Multifunctional Lipid ECO Plasmid DNA Nanoparticles as Efficient Non-viral Gene Therapy for Leber's Congenital Amaurosis. Mol Ther Nucleic Acids 7:42-52
Pyakurel, Aswin; Balmer, Delphine; Saba-El-Leil, Marc K et al. (2017) Loss of Extracellular Signal-Regulated Kinase 1/2 in the Retinal Pigment Epithelium Leads to RPE65 Decrease and Retinal Degeneration. Mol Cell Biol 37:
Sui, Xuewu; Weitz, Andrew C; Farquhar, Erik R et al. (2017) Structure and Spectroscopy of Alkene-Cleaving Dioxygenases Containing an Atypically Coordinated Non-Heme Iron Center. Biochemistry 56:2836-2852
Wu, Lei; Guo, Xin; Hartson, Steven D et al. (2017) Lack of ?, ?-carotene-9', 10'-oxygenase 2 leads to hepatic mitochondrial dysfunction and cellular oxidative stress in mice. Mol Nutr Food Res 61:

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