Browsing by Author "Shisler, Krista Ann"

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Spectroscopic investigations into the active site structure and the mechanisms of radical SAM enzymes
(Montana State University - Bozeman, College of Letters & Science, 2016) Shisler, Krista Ann; Chairperson, Graduate Committee: Joan B. Broaderick; Joan B. Broderick was a co-author of the article, 'Emerging themes in radical SAM chemistry' in the journal 'Current opinion in structural biology' which is contained within this dissertation.; Joan B. Broderick was a co-author of the article, 'Glycyl radical activating enzymes: structure, mechanisms and substrate interactions' in the journal 'Archives of biochemistry and biophysics' which is contained within this dissertation.; Masaki Horitani, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'EPR and ENDOR analysis of small molecules inducing valence localization in PFL-AE' submitted to the journal 'Journal of the American Chemical Society' which is contained within this dissertation.; Rachel U. Hutcheson, Kaitlin S. Duschene, Adam V. Crain, Ashley Rasmussen, Jian Yang, Jessica L. Vey and Joan B. Broderick were co-authors of the article, 'The activation of the radical SAM enzyme pyruvate formate lyase activating enzyme is stimulated by potassium' submitted to the journal 'Biochemistry' which is contained within this dissertation.; Masaki Horitani, Kaitlin Duschene, Rachel U. Hutcheson, Amy Marts, George Cutsail III, William Broderick, Brian M. Hoffman and Joan B. Broderick were co-authors of the article, 'A rapid freeze quench ENDOR study of an organometallic radical intermediate in PFL-AE' submitted to the journal 'Journal of the American Chemical Society' which is contained within this dissertation.; This dissertation contain one article of which Krista Ann Shisler is not the main author.
The radical S-adenosyl-L-methionine (SAM) superfamily of enzymes carry out diverse and complex reactions through generation of a 5'-deoxyadenosyl (5'-dAdo·) radical followed by transfer to substrate. These enzymes contain a [4Fe-4S] cluster which binds and transfers an electron to SAM. The exact mechanism of 5-dAdo· generation is unknown and the studies herein provide further investigation into pyruvate formate lyase activating enzyme (PFL-AE) and lysine 2,3-aminomutase (LAM) pre and post SAM cleavage. To understand the active site of PFL-AE prior to SAM cleavage, cation and small molecule effects were examined by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. Previously, PFL-AE had been observed to contain a valence localized cluster in the presence of small molecules and this work used EPR and ENDOR spectroscopy to further probe the effects of these molecules. These studies determined that these molecules do not directly bind the cluster but rather an H xO species occupies the unique Fe site. The crystal structure of PFL-AE revealed a cation site and to probe this site, EPR and ENDOR spectroscopies were employed. Monovalent cations stimulated PFL-AE activity, with the greatest activity in the presence of potassium. The identity of the cation perturbed the EPR signal of PFL-AE which was more pronounced in the presence of SAM. ENDOR spectroscopy determined that SAM coordination differed depending on the monovalent cation. Due to its high reactivity, 5'-dAdo· has never been spectroscopically observed. In order to examine any intermediate states, a SAM analog and rapid freeze quench (RFQ) techniques were employed in conjunction with EPR and ENDOR spectroscopies. LAM can cleave the SAM analog, S-3',4'-anhydroadenosyl-L-methionine, to produce a stable allylic radical which was coupled with isotopically labeled lysine for ENDOR analysis. It was determined that radical generation is highly controlled with little movement towards its substrate upon 5'-dAdo· production. During RFQ techniques on PFL-AE, an organometallic intermediate species was observed. To probe this intermediate, isotopically labeled SAM and an 57Fe labeled cluster were coupled with the unknown paramagnetic species. It was determined that this intermediate was an unprecedented organometallic Fe-adenosyl bound species post SAM cleavage.