Unraveling the Principles of Catalytic diversity in the Carotenoid Oxygenase Superfamily

Researchers from the University of California Irvine and Stanford University are studying a family of enzymes known as carotenoid cleavage dioxygenases (CCDs) that participate in a broad spectrum of biological processes, including photon detection, hormone synthesis, and lignin degradation. CCDs are classically known to cleave carotenoids, which are colored compounds found abundantly in nature. Additionally, certain CCDs catalyze alternative reactions involving carotenoids, carotenoid derivatives, and non-carotenoid polyenes. Control of CCD function is of interest and importance for a variety of biotechnology-related fields, including agriculture, horticulture, medicine, and biofuel production. Applications in these fields are limited by critical gaps in the understanding of how CCDs can display disparate activities and how these activities could be controlled or modified. The research team will seek an understanding of how CCDs work at the molecular level to achieve their disparate activities. New activity modulators and the ability to rationally alter the structures and activity of CCDs could make inroads in the biotechnological applications of these enzymes. Participation of students, including veterans, in this research and the development of new educational methods will promote their entry and preparation for careers in STEM fields. Carotenoid cleavage dioxygenases are of fundamental biochemical interest due to the diversity of reactions they catalyze using a unique non-heme Fe(II) center coupled with a highly adaptable, substrate-binding pocket. The primary focus of the research will be to elucidate the mechanisms and active-site determinants of the varied activities. The first aim will elucidate molecular details of the CCD-catalyzed oxygenation reaction using stopped-flow absorbance, magnetic circular dichroism (MCD), and other spectroscopic methods. The second aim will apply X-ray crystallography and MCD spectroscopy to advance understanding of the role of iron coordination site occlusion in CCD catalysis and the structural basis for carotenoid substrate stereoselectivity. The third aim will reveal the evolutionary origins of unique CCD activities using ancestral state reconstruction. The fourth aim will provide a structural basis for understanding biosynthesis of strigolactones by CCDs. The proposed studies will advance understanding of CCD evolution and biochemistry, illuminating principles of dioxygen activation and catalytic adaptation. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.