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

科学价值：基因组测序通过实现比较基因组学(系统基因组学)和植物与原核生物研究之间前所未有的信息流，正在改变代谢生物化学的发现。这个拟南芥2010项目利用广泛的生物数据库的力量来发现参与叶酸代谢的新基因的功能。叶酸是一种小的有机分子，它与蛋白质一起充当基本的辅因子，使对所有形式的生命至关重要的酶反应成为可能。叶酸参与的反应是在不同的氧化程度上转移一个碳(C1)单位。尽管几十年来叶酸作为C1代谢的辅助因子进行了经典研究，但系统基因组学揭示了所有生命王国中具有新的叶酸依赖功能的未知蛋白质。这个由系统基因组学驱动的项目旨在确定两个这样的新蛋白质(YgfZ和APE157)在植物和细菌中的功能，阐明第三个蛋白质(TrmE)的功能，并促进系统发育学在代谢生物化学领域的应用。YgfZ蛋白通常是生存所必需的，并且似乎在铁-硫(Fe/S)簇的组装或修复中起作用。一种可行的假设是，YGFZ去除了阻止Fe/S团簇结合位置的C1部分。这一假说将通过细菌和拟南芥的突变、生化和生物物理策略得到验证。遗传学和比较生化证据表明，TrmE蛋白促进了在Fe/S蛋白上增加C1封闭基团。突变和生化方法将被用来检验这一想法。结构和基因组背景信息表明，APE157蛋白通过介导不同的反应来取代关键的叶酸循环酶5-甲酰基四氢叶酸环寡酶(5-FCL)。对于原核生物和植物APE157蛋白，这一假设将通过结合遗传学、代谢物图谱、同位素标记和酶学来检验。将YgfZ定性为参与Fe/S簇代谢的叶酸依赖蛋白，将是叶酸生物化学的新起点，也将建立TrmE、YgfZ和Fe/S酶之间的叶酸联系。确定APE157是5-FCL的广泛替代品在叶酸生物化学中是一个未被探索的方面，具有普遍的重要性。更广泛的影响：尽管系统基因组学是在原核生物和植物中发现未知代谢基因的一种强大且成本效益高的方法，但目前它还没有得到充分利用。为了促进系统基因组学在代谢生物化学中的应用，该项目将利用SEED数据库及其工具开发一个为期两天的讲习班。该研讨会将在获奖期间每年在两所主要大学向目标群体的植物和原核生物化学家介绍，这些人包括博士生、博士后和教职员工。除了这一更广泛的外联活动外，该项目还将有助于在国际和平研究所和共同国际国际实验室培训至少两名博士后和两名博士后，并提供本科生研究机会。通过该项目产生的数据和信息将通过拟南芥信息资源(tair：www.arabidopsis.org)提供。种子库存和任何独特的DNA材料将通过ABRC提供。Http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm

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