Scientific merit: Phylloquinone (vitamin K1) is a fat-soluble molecule that plays a crucial role as electron carrier in photosynthesis. It is synthesized exclusively by plants and certain bacteria. Humans require it as a dietary supply for blood coagulation and for calcium fixation in bones. Although the biosynthesis of phylloquinone has been tacitly assumed to have been defined for more than a decade, the comparative analysis of bacterial and plant genomes reveals that several enzymes involved in phylloquinone biosynthesis are missing. Moreover, plants have evolved an extraordinary metabolic architecture to synthesize this vitamin, including multiple events of gene fusion, highly diverged enzymes, and likely, a separated compartmentalization in chloroplasts and peroxisomes. Plant phylloquinone biosynthesis is also connected via shared intermediates to that of tocopherols (vitamin E), chlorophylls, and salicylate (a plant hormone). Combining comparative genomics, reverse genetics, metabolic profiling, metabolic engineering, and protein biochemistry, this project aims to characterize the missing key steps of phylloquinone biosynthesis, and to establish how plants regulate the flux splits between this pathway and other metabolic branches.
Broader impacts: As plant-based foods represent the main source of dietary vitamin K for humans, this project will raise the awareness of phylloquinone and other plant micronutrient (vitamins, minerals, antioxidants) nutrition for children of the Nebraska 4-H program (Head, Hands, Heart, and Health). For that, the project will develop two presentations and a workshop during which children of middle-school age will learn to calculate how much of each vitamin they get per day during their meals at home and at school, and how these values compare to the recommended dietary intakes. This project will also contribute to the training of two undergraduate research assistants, two PhD students, and one postdoctoral associate in the laboratory of the PI.
Intellectual Merit: The goal of this project was to elucidate the biosynthesis of phylloquinone, a crucial cofactor for photosynthetic organisms and a vitamin (vitamin K1) for vertebrates. We discovered a novel step in the biosynthetic pathway of phylloquinone, and identified the corresponding enzymes in plants and cyanobacteria. We characterized these enzymes using in vitro assays, comparative x-ray diffraction studies (Fig. 1), and metabolic analyses of cognate mutants. The Nomenclature Committee of the International Union of Biochemistry and Molecular Biology used our data to classify these enzymes (EC 3.1.2.28; 1,4-dihydroxy-2-naphthoyl-CoA hydrolase); (www.chem.qmul.ac.uk/iubmb/enzyme/EC3/1/2/28.html). We also provided evidence that in plant cells the biosynthetic pathway of phylloquinone is split between two types of organelles called plastids and peroxisomes (Fig. 2). In parallel, we identified a plant oxidoreductase that connects the formation of disulfide bridges in misfolded proteins to the reduction of phylloquinone, and functionally characterized a multigenic family of enzymes that contribute to the biosynthesis of the terpenoid side-chains of quinones in plastids and mitochondria. Eleven scientific articles resulted from this project and were published in top-tier peer-reviewed journals of international audience. The project also contributed to the training of 3 graduate students and 10 undergraduate students, as well as to the career development of 6 postdoctoral researchers and 2 high-school science teachers. Broader Impact and Outreach: Because plants represent the main source of vitamin K in the western diet, and recent epidemiological studies indicate that suboptimal vitamin K intake is common in the United States, we designed an outreach package aimed at raising awareness about good nutritional habits in children. We ran this educational package to children at the Nebraska 4-H open house to combined group totaling over 200 children. We explained what plant vitamins are and why they are important to our health. We taught the children how to calculate the amount of each vitamin they get at a typical meal at school and at home using simplified spreadsheet from the USDA National Nutrient Database for Standard Reference. Our group also participated to the 'Sunday with a Scientist' outreach program for families on the topic 'Feeding the World in the 21st Century'. In particular, we explained how scientists use biotechnologies to improve plant quality and crop yield (http://newsroom.unl.edu/releases/2011/09/08/Morrill+Hall's+'Sunday+Scientist'+to+explore+plants+Sept.+18+).
