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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Structural and Biophysical Studies of Ribose-5-PhosphateIsomerase A from Francisella TularensisR. Rostankowski 1,2 , M. Orlikowska 1,2 , D. Borek 2 , C. Brautigam 2 ,T. Scheuermann 2 , and Z. Otwinowski 21 Faculty of Chemistry, Department of Medicinal Chemistry, University of Gdansk, Poland;2 Department of Biochemistry, UT Southwestern Medical Center at Dallas, TX, U.S.A.IntroductionRibose-5-phosphate isomerase A (RpiA; EC 5.3.1.6, COG0120) catalyzes theinterconversion of ribose-5-phosphate and ribulose-5-phosphate. This intracellular enzymeis essential in the pentose phosphate pathway and in the Calvin cycle of plants. It isubiquitous and its sequence is highly conserved in species ranging from archaea andbacteria to plants and animals. Whether RpiA is viewed as an anabolic or catabolic enzymedepends on the particular metabolic circumstances. When ribose-5-phosphate is abundant,the reaction runs in the “forward” direction as part of the nonoxidative branch of thepentose phosphate pathway. This ultimately converts the phosphosugar into intermediatesfor glycolysis, and so provides precursors for the synthesis of amino acids, vitamins,nucleotides, and cell wall constituents. In plants, ribulose-5-phosphate is phosphorylated toform ribulose-1,5-bisphosphate, the vital acceptor of CO2 in the first dark reaction ofphotosynthesis. Running “in reverse,” RpiA is the final step in converting glucose-6-phosphate into the ribose-5-phosphate required for synthesis of nucleotides and cofactors;the previous steps, which comprise the oxidative branch of the pentose phosphate pathway,are a major source of the NADPH needed for reductive biosynthesis. Despite its central rolein metabolism, many details of RpiA's action have to date remained poorly understood,largely because of a lack of structural data [1].Results and DiscussionCrystal structure determinationFig. 1. Crystal structure of Ribose-5-phosphate Isomerase A (3KWM).Sitting drop vapor diffusion technique was usedfor the crystallization of RpiA. Structure wassolved using molecular replacement techniquewith substrate and inhibitor complexes of ribose-5-phosphate isomerase A from Vibrio vulnificusYJ016 (3ENQ) as a model. The model was rebuiltin the electron density map. Structure wasdeposited in Protein Data Bank - 3KWM(Figure 1).Hanging drop vapor diffusion method wasused for crystallization of the protein complex withR-5-P. Structure was solved using molecularreplacement method where RpiA (3KWM)structure as a model was used [2].Analytical Ultracentrifugation experimentAUC sedimentation velocity run was carried out at room temperature using a Beckman XLIanalytical ultracentrifuge equipped with UV absorption optics (Beckman Coulter, Brea,CA). Experiment was obtained in conventional double-sector cells. Protein solutioncontained 50 mM HEPES, 200 mM NaCl, 1 mM DDT. The concentrations of protein were0.1 OD, 0.5 OD and 1 OD [3].Isothermal Titration Calorimetry studiesTitrations of ligand into a protein solution were carried out at 30 o C using a VP-ITCinstrument (MicroCal LLC, Northcampton, MA). Both ligand and protein solutionscontained 50 mM pH 7.5HEPES, 50 mM NaCl and 1 mM DTT. The concentrations ofligand and protein were 1mM and 0.05 mM respectively. Experiments were done intriplicate at given temperature and consisted of 25 injections of 10µl (first injection 1 µl).556