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 The reactive form of a C-S bond-cleaving CO 2-fixing flavoenzyme(Montana State University - Bozeman, College of Letters & Science, 2019) Mattice, Jenna Rose; Chairperson, Graduate Committee: Jennifer DuBois; Thesis includes a paper of which Jenna R. Mattice is not the main author.Atmospheric carbon dioxide (CO 2) is used as a carbon source for building biomass in plants and most engineered synthetic microbes. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme on earth, is used by these organisms to catalyze the first step in CO 2 fixation. 1,2 Microbial processes that also fix carbon dioxide or bicarbonate have more recently been discovered. My research focuses on a reaction catalyzed by 2-KPCC (NADPH:2-ketopropyl-coenzyme M oxidorectuase/ carboxylase), a bacterial enzyme that is part of the flavin and cysteine-disulfide containing oxidoreductase family (DSORs) which are best known for reducing metallic or disulfide substrates. 2-KPCC is unique because it breaks a comparatively strong C-S bond, leading to the generation of a reactive enolacetone intermediate which can directly attack and fix CO 2. 2-KPCC contains a phenylalanine in the place where most other DSOR members have a catalytically essential histidine. This research focuses on studying the unique reactive form of 2-KPCC in presence of an active site phenylalanine.Item The coenzyme M biosynthetic pathway in proteobacterium Xanthobacter autotrophicus Py2(Montana State University - Bozeman, College of Letters & Science, 2018) Partovi, Sarah Eve; Chairperson, Graduate Committee: John W. Peters; Florence Mus, Andrew E. Gutknecht, Hunter A. Martinez, Brian P. Tripet, Bernd Markus Lange, Jennifer L. DuBois and John W. Peters were co-authors of the article, 'Coenzyme M biosynthesis in bacteria involves phosphate elimination by a unique member of the aspartase/fumarase superfamily' submitted to the journal 'Journal of biological chemistry' which is contained within this thesis.The metabolically versatile bacterium Xanthobacter autotrophicus Py2 has been the focus of many studies within the field of bioenergy sciences, as it contains two unique CO 2 fixing enzymes, and can utilize unconventional substrates such as propylene and acetone as the sole supplemented carbon source while fixing CO 2 in the process. Unexpectedly, coenzyme M (CoM) was found to play a crucial role as a C 3 carrier in the pathway for propylene metabolism in the late 1990s. Previously, CoM was thought to be present solely as a C 1 carrier in methanogenic archaea for nearly 30 years. Though CoM biosynthesis has been characterized in methanogenic archaea, bacterial CoM biosynthesis remained uncharacterized. In X. autotrophicus Py2, four putative CoM biosynthetic enzymes encoded by xcbB1, C1, D1, and E1 have been identified through informatics and proteomic approaches. XcbB1 is homologous to the archaeal ComA which catalyzes the addition of sulfite to phosphoenolpyruvate, and forms the initial intermediate, phosphosulfolactate, in one of the methanogen CoM biosynthetic pathways. The remaining genes do not encode homologues of any of the previously characterized enzymes in methanogen CoM biosynthesis, suggesting bacteria have a unique pathway. The production of phosphosulfolactate by ComA homolog XcbB1 was verified, indicating that bacterial CoM biosynthesis is initiated in an analogous fashion to the PEP-dependent methanogenic archaeal CoM biosynthesis pathway. XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS), and XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to D-cysteine desulfhydrases. Direct demonstration of activities for XcbB1 and C1 strengthens their hypothetical assignment to a CoM biosynthetic pathway, and puts firm contraints on our proposed functions for XcbD1 and E1. Known AFS members catalyze beta-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. We demonstrate herein that XcbC1 catalyzes a beta-elimination reaction on the substrate phosphosulfolactate to yield sulfoacrylic acid and inorganic phosphate. To our knowledge, beta-elimination reactions releasing phosphate is unprecedented among the AFS, indicating XcbC1 is a unique phosphatase. This work will serve as the framework for future studies aimed at uncovering the final stages of the biosynthetic pathway. By elucidating the XcbB1 and XcbC1 reactions, we have made significant strides towards understanding bacterial CoM biosynthesis which evaded characterization in previous years.