The long term objectives of the research supported by this grant are to determine the novel chemical mechanisms by which vitamin B6 (PLP), Vitamin B12 (adenosylcobalamin), S-adenosylmethionine (SAM), and iron-sulfur clusters function in enzymatic reaction. The conventional role of PLP biology is to stabilize carbanionic intermediates in enzymatic reactions. A major focus of the research supported by this grant is the elucidation of the role of PLP in catalyzing reactions that involve free radical-paramagnetic intermediates. PLP-facilitated radical reactions appear to take place in reactions of aminomutases such as lysine 2,3-aminomutase and lysine 5,6-aminomutase. Radical intermediates of substrates and vitamin coenzymes will be characterized spectroscopically, and the structures of the enzymes that catalyze their reactions will be elucidated. The roles of SAM and [4Fe-4S] clusters in the initiation of radical formation will be unmasked and characterized chemically, spectroscopically, and kinetically. The relationship between the actions of adenosylcobalamin in Vitamin B 12-dependent and SAM/[4Fe-4S]-dependent aminomutases will be explored. Novel chemistry in the actions of vitamins B6 and B12, SAM, and [4Fe-4S] will be discovered. New beta-aminoacids will be produced by engineering of the genes producing aminomutases. Aminomutases are important in amino acid metabolism and the biosynthesis of antibiotics such as Streptothricin F, Myomycin, Blasticidin S, and Taxol. The contributions of beta- amino acids produced by lysine 2,3-aminomutase and arginine 2,3-aminomutase to the functions of antibiotics are not known. The beta-aminoacids may become significant in drug development.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Physical Biochemistry Study Section (PB)
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Sechi, Salvatore
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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Chen, Yung-Han; Maity, Amarendra N; Frey, Perry A et al. (2013) Mechanism-based inhibition reveals transitions between two conformational states in the action of lysine 5,6-aminomutase: a combination of electron paramagnetic resonance spectroscopy, electron nuclear double resonance spectroscopy, and density functional J Am Chem Soc 135:788-94
Chen, Yung-Han; Maity, Amarendra N; Pan, Yu-Chiang et al. (2011) Radical stabilization is crucial in the mechanism of action of lysine 5,6-aminomutase: role of tyrosine-263? as revealed by electron paramagnetic resonance spectroscopy. J Am Chem Soc 133:17152-5
Ruzicka, Frank J; Frey, Perry A (2010) Kinetic and spectroscopic evidence of negative cooperativity in the action of lysine 2,3-aminomutase. J Phys Chem B 114:16118-24
Maity, Amarendra N; Hsieh, Chih-Pin; Huang, Ming-Hui et al. (2009) Evidence for conformational movement and radical mechanism in the reaction of 4-thia-L-lysine with lysine 5,6-aminomutase. J Phys Chem B 113:12161-3
Tang, Kuo-Hsiang; Mansoorabadi, Steven O; Reed, George H et al. (2009) Radical triplets and suicide inhibition in reactions of 4-thia-D- and 4-thia-L-lysine with lysine 5,6-aminomutase. Biochemistry 48:8151-60
Schwartz, Phillip A; Frey, Perry A (2007) 5'-Peroxyadenosine and 5'-peroxyadenosylcobalamin as intermediates in the aerobic photolysis of adenosylcobalamin. Biochemistry 46:7284-92
Ruzicka, Frank J; Frey, Perry A (2007) Glutamate 2,3-aminomutase: a new member of the radical SAM superfamily of enzymes. Biochim Biophys Acta 1774:286-96
Chen, Dawei; Tanem, Justinn; Frey, Perry A (2007) Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase. Biochim Biophys Acta 1774:297-302
Schwartz, Phillip A; Lobrutto, Russell; Reed, George H et al. (2007) Probing interactions from solvent-exchangeable protons and monovalent cations with the 1,2-propanediol-1-yl radical intermediate in the reaction of dioldehydrase. Protein Sci 16:1157-64
Schwartz, Phillip A; Frey, Perry A (2007) Dioldehydrase: an essential role for potassium ion in the homolytic cleavage of the cobalt-carbon bond in adenosylcobalamin. Biochemistry 46:7293-301

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