Browsing by Author "Prussia, Gregory Andrew"

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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.