Arabidopsis 2010: Novel Folate-Related Proteins Shared by Plants and Prokaryotes

University Of Florida, Gainesville FL

Investigators

Abstract

Scientific Merit: Genome sequencing is transforming discovery in metabolic biochemistry by enabling comparative genomics (phylogenomics) and unprecedented information flow between plant and prokaryote research. This Arabidopsis 2010 project uses the power of extensive biological databases to discover the function of new genes involved in folate metabolism. Folates are small, organic molecules that act as essential cofactors in conjunction with proteins to enable enzymatic reactions that are critical to all forms of life. Folates are involved in reactions that transfer one carbon (C1) units at different levels of oxidation. Despite decades of classical research on folates as cofactors in C1 metabolism, phylogenomics reveals unknown proteins in all kingdoms of life that are candidates for novel folate-dependent functions. This phylogenomics-driven project aims to establish the functions of two such novel proteins in plants and bacteria (YgfZ and APE157), to clarify that of a third (TrmE), and to advance use of phylogenomics in the metabolic biochemistry community. YgfZ proteins are often essential to viability and appear to have a role in iron-sulfur (Fe/S) cluster assembly or repair. A working hypothesis is that YgfZ removes C1 moieties blocking Fe/S cluster binding sites. This hypothesis will be tested by mutational, biochemical, and biophysical strategies in bacteria and Arabidopsis. Genetic and comparative biochemical evidence suggests that TrmE proteins facilitate addition of C1 blocking groups to Fe/S proteins. Mutational and biochemical approaches will be used to test this idea. Structural and genomic context information indicates that APE157 proteins functionally replace the crucial folate recycling enzyme 5-formyltetrahydrofolate cycloligase (5-FCL) by mediating a different reaction. For both prokaryotic and plant APE157 proteins, this hypothesis will be tested by combining genetics, metabolite profiling, isotope labeling, and enzymology. Characterizing YgfZ as a ubiquitous folate-dependent protein involved in Fe/S cluster metabolism would be a new departure in folate biochemistry, as would establishing a folate connection between TrmE, YgfZ, and Fe/S enzymes. Identifying APE157 as a widespread alternative to 5-FCL is an unexplored aspect in folate biochemistry of general importance. Broader Impact: Although phylogenomics is a powerful and cost-effective way to discover unknown metabolic genes in prokaryotes and plants it is currently underused. To promote the application of phylogenomics in metabolic biochemistry, this project will develop a two-day workshop using the SEED database and its tools. This workshop will be presented at two major universities per year for the duration of the award to a target group of plant and prokaryotic biochemists, at PhD student, postdoctoral, and faculty levels. In addition to this wider outreach, this project will contribute to the training of at least two PhD students and two postdoctorals in the laboratories of the PI and Co-PI, and provide undergraduate research opportunities. Data and information generated through this project will be made available through the Arabidopsis Information Resource (TAIR: www.arabidopsis.org). Seed stocks and any unique DNA materials will be made available through ABRC. http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm