Theses and Dissertations at Montana State University (MSU)
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Item Delineating the determinants of carboxylation in 2-ketopropyl coenzyme M oxidoreductase/carboxylase: a unique CO 2-fixing flavoenzyme(Montana State University - Bozeman, College of Letters & Science, 2018) Prussia, Gregory Andrew; Chairperson, Graduate Committee: John W. Peters; George H. Gauss, Florence Mus, Leah Conner, Jennifer L. DuBois and John W. Peters were co-authors of the article, 'Substitution of a conserved catalytic dyad causes loss of carboxylation in 2-KPCC' in the journal 'Federation of European Biochemical Societies letters' which is contained within this dissertation.; Jennifer L. DuBois and John W. Peters were co-authors of the article, 'A role for hisitidine 506 in carboxylate stabilization of 2-ketopropyl coenyzme M oxidoreductase/carboxylase' which is contained within this dissertation.; Gregory Andrew Prussia is not the main author of an article which is contained in this dissertation.Global CO 2-emissions are continuously rising, accelerating the impact of associated environmental processes such as climate change, deforestation, and ocean acidification. As a consequence, there is great interest in processes that can mitigate the increase in anthropogenic CO 2. The biological incorporation of a CO 2 molecule into an organic substrate is catalyzed by enzymes known as carboxylases. Although carboxylases employ diverse CO 2-fixing mechanisms and play broad physiological roles in Nature, they follow three general paradigms: 1). The formation of a reactive ene-intermediate nucleophile. 2). Protection of this reactive nucleophile from potential competing electrophiles (other than CO 2) by excluding solvent from the active site. 3). Electrostatic complementation of the negatively-charged carboxylation intermediate and product. 2-ketopropyl coenzyme M oxidoredutase/carboxylase (2-KPCC) is the only known carboxylating member of the FAD-containing, NAD(P)H-dependent disulfide oxidoreductase (DSOR) enzymes. The members of this family catalyze redox reactions and several well-characterized members catalyze the reductive cleavage of disulfide substrate. 2-KPCC performs the reductive cleavage of a thioether bond and subsequently carboxylates it's intermediate. How 2-KPCC has integrated the paradigms of carboxylation using a scaffold purposed for reductive cleavage is unknown. In this work, the paradigms mentioned above are identified in 2-KPCC and the methods by which 2-KPCC integrates carboxylation chemistry with reductive cleavage are discussed. Essential to the redox chemistry catalyzed by many DSOR members is a conserved His-Glu catalytic dyad, which serves to stabilize the electronic interaction between the FAD cofactor and the redox-active cysteine pair in the reactive state. 2-KPCC has substituted the catalytic His and Glu with Phe and His, respectively. We show that the Phe substitution is critical for excluding protons (as competing electrophiles) from the active site and the downstream His substitution acts to stabilize the negative charge on the carboxylated product, acetoacetate. Individually, each substitution plays an essential role in carboxylation. We show through a detailed spectroscopic study that by substituting both catalytic dyad residues the protonated and electronic state of the redox-active cysteine pair and FAD cofactor are affected, altering the DSOR active site to accommodate the unique cleavage and CO 2-fixation reaction catalyzed by 2-KPCC. Thus, this research has furthered the understanding of how the prototypical reductive cleavage reactions catalyzed by DSOR enzymes can be coordinated with a carboxylation reaction by a mechanism analogous to that shared by established carboxylase enzymes.Item Radical S-adenosyl-L-methionine enzymes: radical control and assembly of complex metallocofactors(Montana State University - Bozeman, College of Letters & Science, 2018) Byer, Amanda Shaw; Chairperson, Graduate Committee: Joan B. Broderick; Elizabeth C. McDaniel, Stella Impano, William E. Broderick and Joan B. Broderick were co-authors of the article, 'Mechanistic studies of radical SAM enzymes: pyruvate formate-lyase activating enzyme and lysine 2,3-aminomutase' in the journal 'Methods in enzymology' which is contained within this dissertation.; Masaki Horitani was an author and Krista A. Shisler, Tilak Chandra, Joan B. Broderick and Brian M. Hoffman were co-authors of the article, 'Why nature uses radical S-adenosyl-L-methionine enzymes so widely: electron nuclear double resonance studies of lysine 2,3-aminomutase show the 5'-dADO 'free radical' is never free' in the journal 'Journal of the American Chemical Society' which is contained within this dissertation.; Hao Yang, Elizabeth C. McDaniel, Venkatesian Kathiresan, Stella Impano, Adrien Pagnier, Hope Watts, Carly Denler, Anna Vagstad, Jorn Piel, Kaitlin S. Duschene, Eric M. Shepard, Thomas P. Shields, Lincoln G. Scott, Edward A. Lilla, Kenichi Yokoyama, William E. Broderick, Brian M. Hoffman, and Joan B. Broderick were co-authors of the article, 'New paradigm for radical SAM enzyme reactions: organometallic intermediate Omega is central to catalysis' in the journal 'Journal of the American Chemical Society' which is contained within this dissertation.; Eric M. Shepard was an author and Priyanka Aggarwal, Jeremiah N. Betz, Krista A. Shisler, Robert J. Usselman, Gareth R. Eaton, Sandra S. Eaton, Joan B. Broderick were co-authors of the article, 'Hydrogenase maturase HydF: insights into [2Fe-2S] and [4Fe-4S] cluster communication and hydrogenase activation' in the journal 'Biochemistry' which is contained within this dissertation.; Eric M. Shepard, William E. Broderick and Joan B. Broderick were co-authors of the article, 'Activation of [FeFe]-hydrogenase in the absence of HydG' which is contained within this dissertation.; Donald S. Wright, Michael W. Ratzloff, Yisong Guo, Paul W. King and Joan B. Broderick were co-authors of the article, '[FeFe]-hydrogenase metallocluster assmebly on HydF as influenced by HydG' which is contained within this dissertation.; Amanda Shaw Byer is not the main author of an article which is contained within this dissertation.Electrons, whether from carbon-based radicals or metals, can generate oxidative stress and disease in biological systems; however, when directed properly by a protein, these electrons are responsible for crucial life-sustaining reactions, including photosynthesis, oxygen transport in blood, and nitrogen fixation. Beneficial use of radicals and metallocofactors is abundant in nature, and both are essential in one of the largest superfamilies in biology - the radical SAM (RS) enzyme superfamily. Found in all kingdoms of life, RS enzymes contribute to critical processes such as DNA repair, complex metallocluster assembly, and vitamin synthesis. Understanding how metalloenzymes, such as RS enzymes, control electron flow is critical for comprehending biological system functionality and potentially improving productivity through rational design. This work examines radical control in RS enzyme mechanism and then expands scope to consider RS enzyme contribution to assembly of the complex metallocluster (Hcluster) of [FeFe]-hydrogenase. Focusing in on the fundamental chemistry of RS enzyme radical initiation, this work investigated intermediate states in 5'deoxyadenosyl radical formation by: 1) slowing the radical reaction with a SAM analogue, anSAM, and 2) swiftly stopping catalysis via rapid freeze quench techniques. Employing primarily EPR and ENDOR spectroscopies, two intermediate states were characterized: 1) an analogue of the 5'-deoxyadenosyl radical, formed from anSAM, and 2) an organometallic intermediate, Omega, formed during reaction with SAM. To probe how certain RS enzymes (HydE and HydG) contribute to build the 2Fe H-cluster subcluster precursor on the [FeFe]-hydrogenase scaffold HydF, FeS cluster intermediate states were analyzed using UV-Vis, EPR, FTIR, CD, Mossbauer spectroscopies and gas chromatography. These results demonstrate: 1) HydF initially binds a [4Fe-4S] and a [2Fe-2S] cluster, 2) HydG contributes small molecule diatomics and perturbs the [2Fe-2S] cluster environment, 3) HydE can generate a subcluster precursor on HydF capable of generating catalytically active HydA, and 4) the HydF dimer, not tetramer, delivers the 2Fe H-cluster subcluster precursor for activation. Collectively, this thesis illuminates key mechanistic states RS enzymes use to productively control the 5'deoxyadenosyl radical during catalysis and identifies [FeFe]-hydrogenase H-cluster precursor intermediates suggesting RS enzyme sequentiality.