The SIR provided (I)-Se-Cys; 150mg The ribonucleotide reductase from Lactobacillus leichmannii catalyzed the reduction of NTPs to dNTPs using the cofactor adenosyllcobalamin. Understanding the mechanisms for NTP reduction is important not only because this enzyme is essential for the production of dNTPs and is therefore an important target for drug design, but also because the enzyme is proposed to reduce the NTPs via a novel, radical mechanism. Central to the proposed mechanisms is the formation of an active-site thiyl radical at position Cys 408. To provide evidence for this proposed mechanisms and to understand the role of the thiyl radical in catalysis, this cysteine is being replaced with a variety of amino acids using unnatural amino acid mutagenesis. This methodology allows an amino acid to be synthesized in the lab and then incorporated site-specifically in a protein. Given that the S-H bond strength is similar to that of the substrate C-H bond that is proposed to be homolyzed in the first step of the reduction, Cys 408 is being replaced by amino acids, such as selenocysteine, that are similar in structure to cysteine, but differ in X-H bond strength and kinetic reactivity. Once the amino acids are incorporated into ribonucleotide reductase, the mutant enzymes will be tested for their ability to catalyze the reduction of CTP to dCTP and the exchange of 3H from 3H-adenosyloblamin into 3H2) and may be examined using Electron Spin Resonance Spectroscopy. These replacements should provide insight into the thermodynamic and kinetic requirements for enzyme-catalyzed radical reactions.
| Martinez, Rodolfo A; Glass, David R; Ortiz, Erick G et al. (2014) Synthesis of isotopically labeled 1,3-dithiane. J Labelled Comp Radiopharm 57:338-41 |
| Martinez, Rodolfo A; Glass, David R; Ortiz, Erick G et al. (2013) Large-scale preparation of (13) C-labeled 2-(phenylthio)acetic acid and the corresponding labeled sulfoxides and sulfones. J Labelled Comp Radiopharm 56:31-5 |
| Creager, Melinda S; Jenkins, Janelle E; Thagard-Yeaman, Leigh A et al. (2010) Solid-state NMR comparison of various spiders' dragline silk fiber. Biomacromolecules 11:2039-43 |
| Jenkins, Janelle E; Creager, Melinda S; Lewis, Randolph V et al. (2010) Quantitative Correlation between the protein primary sequences and secondary structures in spider dragline silks. Biomacromolecules 11:192-200 |
| Kim, Sun Hee; Aznar, Constantino; Brynda, Marcin et al. (2004) An EPR, ESEEM, structural NMR, and DFT study of a synthetic model for the covalently ring-linked tyrosine-histidine structure in the heme-copper oxidases. J Am Chem Soc 126:2328-38 |
| Ollivault-Shiflett, Morgane; Kimball, David B; Silks, L A Pete (2004) Synthesis of chiral 13C,77Se-labeled selones. J Org Chem 69:5150-2 |
| Schmidt, Bryan; Hillier, Warwick; McCracken, John et al. (2004) The use of stable isotopes and spectroscopy to investigate the energy transducing function of cytochrome c oxidase. Biochim Biophys Acta 1655:248-55 |
| Schmidt, Bryan; McCracken, John; Ferguson-Miller, Shelagh (2003) A discrete water exit pathway in the membrane protein cytochrome c oxidase. Proc Natl Acad Sci U S A 100:15539-42 |
| Gray, Chandele R; Sanz-Cervera, Juan F; Silks, Louis A et al. (2003) Studies on the biosynthesis of asperparaline A: origin of the spirosuccinimde ring system. J Am Chem Soc 125:14692-3 |
| Van Dien, Stephen J; Strovas, Tim; Lidstrom, Mary E (2003) Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using 13C-label tracing and mass spectrometry. Biotechnol Bioeng 84:45-55 |
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