Osmotic Pressure and Crowding Effects on Folate Mediated One Carbon Metabolism

描述（由申请人提供）：Howell实验室先前的研究发现，将中性渗透物添加到r质粒编码的二氢叶酸还原酶（R67 DHFR）以及非同源大肠杆菌染色体DHFR （EcDHFR）中会导致底物二氢叶酸（DHF）的结合较弱。这一结果与相同条件下NADPH的紧密结合形成对比。辅助因子的结果更典型，因为减少的水含量减少了与结合相关的脱溶惩罚。一个解释DHF与DHFR结合较弱的模型假设，二氢叶酸与各种渗透物之间存在弱相互作用。如果DHF“渗透物”的相互作用比DHF“水”的相互作用更强，则DHF“渗透物”对失去的能量更多，DHF与DHFR结合的难度更大。（换句话说，与叶酸相比，水更倾向于与渗透物相互作用。）为了拓展酶作用的溶剂替代假说，我们将研究不同的叶酸衍生物以及不同的叶酸利用酶。目的1提出了一系列实验来研究DHF和叶酸衍生物如何与渗透物相互作用。汉斯图将用于确定疏水性是否是使叶酸具有“粘性”的特征元素。目的2将这些生化研究扩展到包括碳代谢中的其他酶。Aim 3回到DHFR，研究大分子拥挤对DHFR活性的影响，因为在渗透物中发现的基序也可以在蛋白质中找到，从而提出DHF“拥挤相互作用”会发生并影响催化效率。Aim 4提出了一系列在大肠杆菌低水活度条件下通过基因互补试验进行叶酸“渗透物”相互作用模型的体内试验。据预测，渗透胁迫会导致参与叶酸介导的一碳代谢的酶的催化效率降低，最终导致生长受阻。Aim 4B考虑渗透物对DHFR对宿主大肠杆菌产生的抗菌耐药性的影响，并讨论DHF和DHFR浓度的变化。这项开创性的研究确定了渗透压（或低水活度）是调节DHFR功能的关键环境因素。由此推而广之，其他重要的叶酸利用酶也会受到影响。影响叶酸或维生素B9浓度、半衰期和可及性的因素对人类健康很重要，因为叶酸的氧化还原形式是单碳循环中的底物和辅酶，影响氨基酸和核酸代谢。由于动物不能合成叶酸或维生素B9，它们必须从饮食中获取。叶酸营养不足的后果可以在怀孕的不良后果中看到，因此最近的叶酸强化食品。抗叶酸也被用来治疗细菌感染、疟疾、癌症、关节炎、心血管疾病等。这些是叶酸生物化学在人体健康中发挥作用的几个例子。

DESCRIPTION (provided by applicant): Previous studies in the Howell lab have found that addition of neutral osmolytes to an R-plasmid encoded dihydrofolate reductase (R67 DHFR) as well as the non-homologous E. coli chromosomal DHFR (EcDHFR) result in weaker binding of the substrate, dihydrofolate (DHF). This result contrasts with the tighter binding of NADPH under these same conditions. The cofactor results are more typical, as the decreased water content reduces the desolvation penalty associated with binding. A model to explain the weaker binding of DHF to DHFR hypothesizes that weak interactions occur between dihydrofolate and various osmolytes. If the DHF"osmolyte interaction is stronger than the DHF"water interaction, then it will take more energy to lose the DHF"osmolyte pair and binding of DHF to DHFR will be more difficult. (In other words, water prefers to interact with osmolytes as compared to folate.) To expand this solvent substitution hypothesis of enzyme action, different folate derivatives as well as different folate utilizing enzymes will be studied. Aim 1 proposes a series of experiments to study how DHF and folate derivatives interact with osmolytes. A Hansch plot will be used to determine if hydrophobicity is the signature element that makes folate "sticky." Aim 2 extends these biochemical studies to include other enzymes in one carbon metabolism. Aim 3 returns to DHFR to study the effects of macromolecular crowding on DHFR activity as the motifs found in osmolytes can also be found in proteins, leading to the proposal that DHF"crowder interactions will occur and affect catalytic efficiency. Aim 4 proposes a series of in vivo tests of the folate"osmolyte interaction model by genetic complementation assays performed under low water activity conditions in E. coli. Osmotic stress is predicted to result in lower catalytic efficiencies for enzymes involved in folate mediated one carbon metabolism, ultimately leading to blockage of growth. Aim 4B considers osmolyte effects on antibacterial resistance conferred by DHFR upon host E. coli and addresses what happens to the DHF and DHFR concentrations. This groundbreaking research identifies osmolality (or low water activity) as a key environmental factor involved in modulating DHFR function. By extension, other important folate utilizing enzymes will also be impacted. Factors that affect the folate, or vitamin B9, concentration, half-life and accessibility are important to human health as the redox forms of folate are substrates and coenzymes in the one carbon cycle, which impacts amino acid and nucleic acid metabolism. As animals do not synthesize folate or vitamin B9, they must obtain it from their diet. The consequences of insufficient folate nutrition can be seen in the adverse outcomes of pregnancy, thus the recent folate fortification of foods. Antifolates are also used as treatments o bacterial infections, malaria, cancer, arthritis, cardiovascular disease, etc. These are a few examples where folate biochemistry plays a role in human health.