Theses and Dissertations at Montana State University (MSU)
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Item A molecular, structural, and cellular multiple-level study aimed at understanding the unique reaction catalyzed by the last enzyme in the heme-biosynthesis pathway of gram-positive bacteria, coproheme decarboxylase (CHDC)(Montana State University - Bozeman, College of Letters & Science, 2018) Celis Luna, Arianna I.; Chairperson, Graduate Committee: Jennifer DuBois; Bennett R. Streit, Garrett C. Moraski, Timothy D. Lash, Gudrun S. Lukat-Rodgers, Kenton R. Rodgers and Jennifer L. DuBois were co-authors of the article, 'Unusual peroxide-dependent, heme-transforming reaction catalyzed by hemQ' in the journal 'Biochemistry' which is contained within this thesis.; George H. Gauss, Bennett R. Streit, Krista Shisler, Garrett C. Moraski, Kenton R. Rodgers, Gudrun S. Lukat-Rodgers, John W. Peters and Jennifer L. DuBois were co-authors of the article, 'A structure-based mechanism for oxidative decarboxylation reactions mediated by amino acids and heme propionates in coproheme decarboxylase (hemQ)' in the journal 'Journal of the American Chemical Society' which is contained within this thesis.; Dissertation contains two articles of which Arianna I. Celis Luna is not the main author.Heme b is one of nature's most ancient and versatile co-factors and is essential for aerobic life. As such, heme b is synthesized by almost every living organism and plays a major role in bacterial virulence. A pathway for heme b biosynthesis, which is unique to some of the most primitive gram-positive bacteria including many important pathogens, was recently discovered. This pathway, now known as the coproprophyrin-dependent (CPD) branch, ends in a step catalyzed by an unusual enzyme known alternately as coproheme decarboxylase (ChdC) or HemQ. This research aimed to understand ChdC function at the molecular, structural, and cellular levels. Using the ChdC enzyme from Staphylococcus aureus (SaChdC) and a variety of biochemical and analytical tools (conventional and stopped-flow UV-Vis spectroscopy, resonance Raman, HPLC, LC-MS, site-directed mutagenesis, EPR, and X-ray crystallography), the work presented here describes how the coproheme substrate is accommodated in the SaChdC active site and poised for reactivity. The cumulative results show that ChdC catalyzes the oxidative decarboxylation of coproheme III to generate heme b in a sequential and clock-wise fashion, generating harderoheme III in the process. This reaction is H 2 O 2-dependent and the mechanism involves the formation of the high-valent Fe(IV) intermediate (Compound I) and a tyrosine radical (Tyr °). The coproheme-bound ChdC structure revealed a helical-loop that is flexible and moves in towards the active site in the presence of substrate. This loop is hypothesized to act as an 'active site gate' which mediates substrate entry and product egress. Due to the cytotoxicity of heme and its porphyrin precursors, we proposed that the metabolite flux in this pathway is controlled by transient protein-protein interactions. Using the UV-Vis characteristics of porphyrins and phenotype characterization of the deltachdC knock-out strain of S. aureus complemented with ChdC point mutants, we present preliminary evidence for an interaction between ChdC the preceding enzyme of the pathway, CpfC. The same approaches also implicated potential interactions between ChdC and an unidentified heme-chaperone, which delivers heme to its final cellular destination. We propose that this chaperone is HemW. Experiments to test this hypothesis are outlined. This work elucidates yet different way that nature has equipped cells to perform radical chemistry in order to accomplish essential molecule transformations, such as that of decarboxylation and the simultaneous generation of CO 2, and emphasizes the importance of substrate/product post-catalysis cellular trafficking.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.Item Mechanism of diatomic ligand biosynthesis by radical s-adenosylmethionine [FeFe]-hydrogenase maturase HydG(Montana State University - Bozeman, College of Letters & Science, 2014) Duffus, Benjamin Richard; Shourjo Ghose, John W. Peters and Joan B. Broderick were co-authors of the article, 'Reversible H atom abstraction at the tyrosine phenol position catalyzed by the radical SAM enzyme HydG' submitted to the journal 'Journal of the American Chemical Society' which is contained within this thesis.; Simon J. George, Aubrey D. Scott, Eric M. Shepard, Kaitlin D. Duschene, Stephen P. Cramer, John W. Peters and Joan B. Broderick were co-authors of the article, 'Defining a basis for diatomic ligand product binding to the radical SAM enzyme HydG' submitted to the journal 'Biochemistry' which is contained within this thesis.; Rebecca C. Driesener, Eric M. Shepard, Peter L. Roach, John W. Peters and Joan B. Broderick were co-authors of the article, 'HydG carbon monoxide formation stoichiometry: the role of phosphate in diatomic ligand biosynthesis' submitted to the journal 'Biochemistry' which is contained within this thesis.; Eric M. Shepard, John W. Peters and Joan B. Broderick were co-authors of the article, 'Effector and intermediate molecule interaction with radical SAM [FeFe]-hydrogenase maturase HydG' submitted to the journal 'Biochemistry' which is contained within this thesis.; Eric M. Shepard, John W. Peters and Joan B. Broderick were co-authors of the article, 'Delineating H atom abstraction in HydG catalysis with tyrosine analogues and site-directed mutagenesis' submitted to the journal 'Biochemistry' which is contained within this thesis.Iron-sulfur (Fe-S) clusters are ubiquitous in biology, and serve as catalysts in a vast array of chemical transformations that comprise central metabolic reactions and small molecule interconversions. Complex Fe-S clusters such as the [FeFe]-hydrogenase "Hcluster" cofactor are part of a distinct subgroup of metalloenzymes that have evolved from reduced Fe-S mineral phases, as the H-cluster catalyzes H-H bond formation through reduction of protons with electrons. Biosynthesis of this cofactor is unique in its involvement of two radical S-adenosylmethionine (SAM) enzymes HydG and HydE, and a scaffold GTPase HydF. Together, these proteins synthesize a unique Fe-S cluster that coordinates a bridging dithiolate ligand as well as two CN- and three CO ligands However, many mechanistic details relating to the biosynthesis are not well known. In this work, the radical SAM enzyme HydG has been shown to synthesize CO, CN-, and pcresol through a radical-initiated fragmentation of the substrate tyrosine. The catalytic mechanism is complex, because an accessory C-terminal Fe-S cluster is required for catalysis. The exact role of this cluster in the biosynthetic mechanism is unresolved, but is proposed to serve a modular role as a potential scaffold for diatomic ligand synthesis. To understand the catalytic mechanism, a combined biochemical and spectroscopic approach was applied. In this work, it is shown that the C-terminal Fe-S cluster is essential for the formation of both CO and CN- products. Spectral characterization of the enzyme has shown the formation of diatomic ligand products that are bound to the coordinated Fe-S clusters. Also, an H atom abstraction profile of HydG has been recently characterized to provide insight to the involvement of the 5'-deoxyadenosyl radical in catalysis. Further mechanistic insight into catalysis has also been investigated through site-directed mutagenesis and through using substrate analogues. The work presented as a whole, by establishing parallels to the radical SAM enzyme superfamily in character to biosynthesis, reveals unifying themes in complex metal cluster assembly related to radical-initiated modification of ordinary Fe-S clusters via product organometallic complex formation.Item Biosynthesis of hydrocarbons in the American cockroach, Periplaneta americana(Montana State University - Bozeman, College of Letters & Science, 1971) Conrad, Charles WaylandItem The synthesis of disaccharides for the functionalization of PAMAM dendrimers(Montana State University - Bozeman, College of Letters & Science, 2011) Nissen, Shannon Rae; Chairperson, Graduate Committee: Mary J. CloningerAn increase in the number of incidences of fungal disease among immuno-compromised patients has developed in recent years. While a patient with an intact immune system would be able to fight the pathogen, an immunocompromised patient is not able to do so. The mortality rate of disseminated candidiasis can be as high as 30-40%. Although there have been great advances in the understanding of how the immune system detects pathogens, there is still much to be learned. PAMAM (poly(amidoamine)) dendrimers have been chosen as scaffolds on which to display disaccharides that are found on the surface of Candida albicans. Preliminary results from immunostimulation assays using a(1,2)-dimannose functionalized PAMAM dendrimers showed that disaccharide functionalized dendrimers can stimulate cytokine production. In light of these results, several disaccharides - phenyl 2-O-benzyl-4,6-O-benzylidene-3-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-1-thio-a-D-mannopyranoside, 1-O-2-(2-azidoethoxy)ethyl-3-O-benzyl-4,6-O-benzylidene-2-O-(2,3-di-O-benzyl-4,6-O-benzylidene-B-D-mannopyranosyl)-a-D-mannopyranoside, 1-O-2-(2-azidoethoxy)ethyl-3-O-benzyl-4,6-O-benzylidene-2-O-(2,3-di-O-benzyl-4,6-O-benzylidene-B-D-glucopyranosyl)-B-D-glucopyranoside, 1-O-2-(2-azidoethoxy)ethyl-2-O-benzyl-4,6-O-benzylidene-3-O-(2,3-di-O-benzyl-4,6-O-benzylidene-B-D-glucopyranosyl)-B-D-glucopyranoside - have been synthesized for the functionalization of G(3.5) PAMAM dendrimers.Item Biochemical, spectroscopic, and structural investigations on [FeFe]-hydrogenase maturation and complex metallocluster assembly(Montana State University - Bozeman, College of Letters & Science, 2010) Mulder, David Wayne; Chairperson, Graduate Committee: John W. PetersMetals are present in nearly half of all enzymes, often at the active site, where they modulate catalytic function. Some of these metalloenzymes exist with a single bound metal ion while many others contain complex metal clusters. Complex FeS assemblies are associated with the interconversion of the small molecules H 2, CO, CO 2, N 2, and NH 3. One such complex metalloenzyme, [FeFe]-hydrogenase, catalyzes the reversible oxidation of molecular H 2. The active site of [FeFe]-hydrogenases, the Hcluster, exists as a [4Fe-4S]-subcluster bridged by a protein thiolate ligand to a 2Fesubcluster which contains biologically unique CO and CN- ligands and a dithiolate ligand. The H-cluster is synthesized by the activities of the hydrogenase maturation enzymes HydE, HydF, and HydG and until recently little was known concerning the biosynthetic pathway for the H-cluster. The results presented here provide significant insight into the stepwise mechanism of H-cluster biosynthesis. Biochemical and spectroscopic characterization of the structural [FeFe]-hydrogenase enzyme expressed in a genetic background devoid of maturation genes hydE, hydF, and hydG (HydA Delta EFG) indicates by the presence of a [4Fe-4S] cluster required for [FeFe]-hydrogenase activation that the [4Fe-4S]-subcluster and 2Fe-subcluster of the H-cluster are synthesized independently. The determination of the x-ray crystal structure of HydA Delta EFG confirms this by revealing the presence of the [4Fe-4S]-subcluster and an open binding pocket for the 2Fe-subcluster, indicating that H-cluster synthesis is directed in a stepwise manner with synthesis and insertion of the [4Fe-4S]-subcluster occurring first by generalized host cell machinery followed by synthesis and insertion of the 2Fe-subcluster by specialized hyd encoded maturation machinery. The structure also reveals that insertion of the 2Fe-subcluster occurs through a positively charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. By utilizing complementary gene data base searching with these structural studies, new insight is made known into the evolutionarily relationships between [FeFe]-hydrogenases present in microorganisms and the eukaryotic Nar1 family of proteins which function in iron-sulfur cluster biosynthesis. The work presented as a whole, by establishing parallels to complex metal cofactor biosynthesis in nitrogenase, reveals unifying themes in complex metal cluster assembly and fundamental features of metalloenzyme evolution.Item Protein cage architectures as a nano-platform for material synthesis and metal binding(Montana State University - Bozeman, College of Letters & Science, 2006) Allen, Mark Andrew; Chairperson, Graduate Committee: Trevor DouglasSupramolecular proteins that assemble into cage like architectures have been used for nano-material synthesis and as a scaffold for metal binding. Material synthesis can be performed by exploiting the cage-like properties of these nano-containers and relying on the electrostatically distinct interior environment that drive mineral encapsulation. Ferritin and ferritin like proteins can be used as size constrained reaction vessels that encapsulate materials that have sizes that are determined by the internal dimensions of the protein cage. These range from 5 nm for the ferritin like protein from Listeria innocua to 24 nm for the interior of an engineered plant virus (Cowpea chlorotic mottle virus). Inorganic materials synthesized within these constrained reaction volumes are monodisperse in size. The crystallinity and phase of material prepared is determined by the reaction conditions, which are mild compared to other preparative methods.Item Investigating the role of iron sulfur cluster binding residues of HYDF(Montana State University - Bozeman, College of Letters & Science, 2012) Joshi, Neelambari; Chairperson, Graduate Committee: John W. Peters[FeFe]-hydrogenases are metalloenzymes found in many bacteria and lower eukaryotes. The catalytic active site of [FeFe]-hydrogenases termed as H-cluster consists of a [4Fe-4S] cubane bridged to a 2Fe subcluster. The two iron atoms of the 2Fe subcluster are decorated by carbon monoxide, cyanide ligands as well as a bridging dithiolate ligand. The assembly of this complex H cluster involves the role of three accessory enzymes namely HydE, HydG and HydF. The maturase, HydF is a GTPase and contains two types of clusters, a [4Fe-4S] and a [2Fe-2S] cluster. The [2Fe-2S] cluster is transformed into an H-cluster precursor by action of HydE and HydG. It is suggested from EPR spectroscopic data of both reduced HydF DeltaEG and Oxidized HydF EG that the [2Fe-2S] cluster and the [4Fe-4S] cluster are not bound to each other. Since an H-cluster like signal was observed in oxidized HydF EG suggested that the two clusters are arranged in same manner as the H-cluster itself. This aforementioned hypothesis drove us to investigate the ligand arrangement of both a [4Fe-4S] and most importantly the [2Fe-2S] clusters in HydF. The apo HydF structure does not provide us with significant insights into Fe-S cluster coordination details, therefore we have attempted to experimentally identify the residues that act as ligands to both the clusters. To that end, we substituted each of the conserved Fe-S cluster binding residues and observed the effects of these mutations on both clusters by spectroscopic methods like UV-Vis spectroscopy and EPR. Our observations indicated that among the three conserved cysteines, C304 and C356 are absolutely quintessential for iron sulfur cluster assembly in HydF DeltaEG while C353 and H306 have some capacity to bind iron sulfur clusters. Further in vitro hydrogenase assays suggested importance of C353 residue as it affected the assembly of the 2Fe subcluster. Thus we propose a dimeric/ tetrameric model of HydF where both the [2Fe-2S] and the [4Fe-4S] clusters are ligated by eight conserved, four putative Fe-S cluster binding residues from each monomer. In our proposed model we discuss the possible occurrence of non cysteinyl ligation for iron sulfur clusters.