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Dissertation-CdelaSena-OSBP2014.pdf (4.46 MB)

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Substrate specificity and reaction mechanism of vertebrate carotenoid cleavage oxygenases

dela Seña, Carlo C
http://rave.ohiolink.edu/etdc/view?acc_num=osu1396444100

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Carotenoids are yellow, orange and red pigments found in fruits and vegetables. Some carotenoids can act as dietary precursors of vitamin A. Humans and other animals generate retinal (vitamin A aldehyde) from provitamin A carotenoids by oxidative cleavage of the central 15-15′ double bond by the enzyme β-carotene 15-15′-oxygenase (BCO1). Another carotenoid oxygenase, β-carotene 9′-10′-oxygenase (BCO2), catalyzes the oxidative cleavage of the 9′-10′ double bond of various carotenoids to yield apo-10′-carotenals and ionones. In this dissertation, we elucidate the substrate specificity of these two enzymes. Recombinant His-tagged human BCO1 was expressed in Escherichia coli strain BL21-Gold (DE3) and purified by cobalt ion affinity chromatography. The enzyme was incubated with various dietary carotenoids and β-apocarotenals, and the reaction products were analyzed by reverse-phase high-performance liquid chromatography (HPLC). We found that BCO1 catalyzes the oxidative cleavage of only provitamin A dietary carotenoids and β-apocarotenals specifically at the 15-15′ double bond to yield retinal. A notable exception is lycopene, which is cleaved by BCO1 to yield two molecules of acycloretinal. Previous studies have found lycopene to be unreactive with BCO1. Our results warrant a fresh look at acycloretinal and its alcohol and acid forms as possible metabolites of lycopene. We also found that BCO1 does not react with 9-cis-β-carotene. It has been previously suggested the 9-cis-retinoic acid, a ligand of retinoid X receptors (RXR's), is generated by BCO1 cleavage of 9-cis-β-carotene to 9-cis-retinal and subsequently oxidized to the acid. However, our results strongly argue against this. Similarly, the substrate specificity of purified recombinant chicken BCO2 was also tested by incubating purified enzyme with the test substrates and analysis of the products by HPLC. Unlike BCO1, BCO2 reacts with full length provitamin A carotenoids as well as non-provitamin A carotenoids that contain 3-hydroxy ionone rings. However, it does not react with lycopene and β-apocarotenals. Carotenoid cleavage oxygenases (CCO's) oxidatively cleave carotenoids to yield aldehydes and/or ketones. Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase based on a study that used conditions that favored oxygen exchange with water. We elucidated the reaction mechanism of BCO1 using the same principles of oxygen labeling experiments, but minimized the reaction and processing times to minimize oxygen exchange between retinal and water. We incubated purified recombinant human BCO1 and β-carotene in an 16O2-H218O medium for 15 minutes at 37°C, and the relative amounts of 18O-retinal and 16O-retinal were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The 18O-retinal makes up only 3-10% of the total retinal. Incubation of 16O-retinal under the same conditions yields 5-13% 18O-retinal. We also incubated BCO1 and β-carotene in an 18O2-H216O medium. Under these conditions, 18O-retinal makes up 79-85% of the total retinal product. Similarly, incubation of 91% 18O-retinal under the same conditions yields 67-84% 18O-retinal. Our results show that BCO1 incorporates only oxygen from O2 into retinal, and BCO1 is therefore a dioxygenase.
Earl Harrison (Advisor)
Robert Curley, Jr. (Committee Member)
Steven Schwartz (Committee Member)
Ross Dalbey (Committee Member)
138 p.
Biochemistry; Nutrition
Carotenoid; vitamin A; metabolism; kinetics; retinal; acycloretinal; lycopene; apocarotenal; enzyme mechanisms; enzyme catalysis; oxidation-reduction; oxygenase; monooxygenase; dioxygenase

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Citations

  • dela Seña, C. C. (2014). Substrate specificity and reaction mechanism of vertebrate carotenoid cleavage oxygenases [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1396444100

    APA Style (7th edition)

  • dela Seña, Carlo. Substrate specificity and reaction mechanism of vertebrate carotenoid cleavage oxygenases. 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1396444100.

    MLA Style (8th edition)

  • dela Seña, Carlo. "Substrate specificity and reaction mechanism of vertebrate carotenoid cleavage oxygenases." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1396444100

    Chicago Manual of Style (17th edition)

osu1396444100
730
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