A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (Coenzyme Q) in Arabidopsis

Authors: LATIMER, SCOTT - University Of Florida; KEENE, SHEA - University Of Florida; BERGER, ANTOINE - University Of Florida; BERNET, ANN - University Of Florida; SOUBEYRAND, ERIC - University Of Florida; WRIGHT, JANET - University Of Nebraska; CLARKE, CATHERINE - University Of California (UCLA); Block, Anna; COLQUHOUN, THOMAS - University Of Florida; ELOWSKY, CHRISTIAN - University Of Nebraska; CHRISTENSEN, ALAN - University Of Nebraska; BASSET, GILLES - University Of Florida

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/4/2021
Publication Date: 10/6/2021
Citation: Latimer, S., Keene, S.A., Berger, A., Bernet, A., Soubeyrand, E., Wright, J., Clarke, C.F., Block, A.K., Colquhoun, T.A., Elowsky, C., Christensen, A., Basset, G.J. 2021. A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (Coenzyme Q) in Arabidopsis. Journal of Biological Chemistry. 297,101283. https://doi.org/10.1016/j.jbc.2021.101283.
DOI: https://doi.org/10.1016/j.jbc.2021.101283

Interpretive Summary: It is widely known that vitamins are important nutrients for human and animal health, yet these are not the only compounds vital for life. Plants and animals also produce several other compounds that are absolutely necessary for survival. One such compound is the vital co-factor Coenzyme Q (CoQ), and lack of CoQ leads to death in plants and animals during embryo development. In addition, its antioxidant nature means that CoQ accumulation also protects against various environmental stresses, fueling an interest in both dietary CoQ supplementation and plant breeding for elevated CoQ content. Surprisingly, despite its importance, it is still not fully known how plants make CoQ. In this study ARS scientists at the Center for Medical, Agricultural, and Veterinary Entomology in Gainesville, FL, in collaboration with researchers from the University of Florida, the University of Nebraska-Lincoln, and the University of California-Los Angeles, address this knowledge gap by uncovering the missing gene responsible for the penultimate step in CoQ synthesis in plants. This research brings us one step closer to understanding how this important compound is made, and thus being able to use knowledge guided strategies to enhance its production in crops.

Technical Abstract: It is not known how plants catalyze the C-6 hydroxylation of demethoxyubiquinone, the penultimate step in the biosynthesis of the vital respiratory cofactor and liposoluble antioxidant, ubiquinone (Coenzyme Q). Combining searches of embryo-defective mutant databases in Arabidopsis thaliana with cross-species gene network modeling, we identified embryo lethal locus EMB2421 (At1g24340) as a top candidate for the missing plant demethoxyubiquinone hydroxylase. Unlike the prototypical eukaryotic demethoxyubiquinone hydroxylase, which is a carboxylate-bridged diiron monooxygenase, At1g24340 is homologous to FAD-dependent oxidoreductases that use NAD(P)H as an electron donor. Complementation assays in yeast and Escherichia coli demonstrated that At1g24340 encodes a functional demethoxyubiquinone hydroxylase, and that the enzyme displays strict specificity for the C-6 position of the benzoquinone ring. Laser-scanning confocal microscopy indicated that GFP-tagged At1g24340 is targeted to mitochondria. Silencing of At1g24340 resulted in up to 74% decrease in ubiquinone content and de novo biosynthesis. Consistent with the role of At1g24340 as a benzenoid ring modification enzyme, such a metabolic blockage could not be by-passed by supplementation with 4-hydroxybenzoate, the immediate precursor of ubiquinone's ring. Isotopic feeding assays in At1g24340-overexpressing transgenics showed that, unlike the situation observed in yeast, the hydroxylation of demethoxyubiquinone exerts limited control over ubiquinone biosynthetic flux in plants. Phylogenetic reconstructions indicated that plant demethoxyubiquinone hydroxylase is most closely related to prokaryotic monooxygenases that act on halogenated aromatics, and likely descends from an event of horizontal gene transfer between a green alga and a bacterium.