Theses and Dissertations at Montana State University (MSU)
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/732
Browse
14 results
Search Results
Item The roles of Phospholipase C in oil biosynthesis in oilseeds(Montana State University - Bozeman, College of Agriculture, 2018) Aryal, Niranjan; Chairperson, Graduate Committee: Chaofu LuResearch on the biosynthesis pathways of storage oil (triacylglycerol, or TAG) in plants has gained momentum for the last couple of decades. Despite significant achievements, a complete understanding of such pathways is still lacking. Also, the production of industrially important unusual oils such as hydroxy fatty acids (HFA) at the commercial level is limited in both native and transgenic plants. In this study, I have examined the roles of Phospholipase C in the TAG biosynthesis. To test roles of non-specific PLCs (NPCs) in TAG synthesis, FA composition was analyzed in mature seeds of T-DNA lines of 6 Arabidopsis npc T-DNA mutants. Among them, npc1, npc4 and npc6 showed increases in oleic acid (18:1) and decreases in lineoleic acid (18:2) and linolenic acid (18:3). The 18:1 was further increased in the npc1/npc6 double mutant compared to npc1 and npc6. These changes in FA profile suggest the role of NPCs in TAG biosynthesis. Given the possible roles of NPCs in TAG synthesis in the seeds of Arabidopsis, I searched for suitable PLC from Castor (RcPLC) for further investigation and to transform to the 7-1 line of Camelina sativa expressing a fatty acid hydroxylase (RcFAH12). From previously published Castor transcriptome, RcPLCL1 was the phospholipase C-like gene that was primarily expressed in the endosperm. Exploration of the RcPLCL1 protein using bioinformatics tools placed it to the PLC-like phosphodiesterase family which has not been characterized yet. Based on the sequence analyses, the protein contains the X domain for the PLC activity on phosphatidylinositols (PI), but it lacks the Y and C2 domains. Enzyme assays using heterologous expression in yeast showed that the protein had both PC- and PI-PLC activities. Based on the genomic sequence analysis, 8 genes were found in Arabidopsis that were predicted to encode PLC-like phosphodiesterases. The seeds of 5 T-DNA knockout lines were analyzed for fatty acid composition, which showed decreased linolenic acid (18:3) and increased oleic acid (18:1). The FA composition change suggested the involvement of these proteins in oil biosynthesis in Arabidopsis seeds. Upon transformation of RcPLCL1 into the 7-1 line, HFA content in the seeds was increased to as high as 24% from 15%. Concurrently the amount of HFA decreased in the membrane lipid phosphatidylcholine (PC). However, the Arabidopsis homologue, AtPLCL1, did not increase the HFA in seeds of 7-1. These results suggest that RcPLCL1 is involved in HFA accumulation. The HFA-producing 7-1 plants were tested in the field. Results from two-year experiments indicated that both wild type and 7-1 demonstrated better performance for traits like oil content and yield. However, the HFA level in 7-1 was significantly decreased in field-grown plants than greenhouse plants.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 Effect of the WRKY76 transcription factor on starch biosynthesis and plant growth(Montana State University - Bozeman, College of Agriculture, 2016) Estabrooks, Hannah Margaret; Chairperson, Graduate Committee: Jack MartinStarch is an important contributor to plant growth as excess photosynthate is stored in leaves as starch during the day to be mobilized at night and re-synthesized in sink tissues. Starch is also the principal constituent of cereal seeds and its variation greatly influence crop yields. The starch pathway is complex and its regulation is not fully understood. Transcription Factors (TFs) are known to act as master regulators of whole biosynthetic pathways and the work presented here was aimed at gaining a better understanding of starch production in leaves by identifying a TF which specifically regulates the leaf starch biosynthetic pathway. Leaf starch levels are regulated in part by ADP-glucose pyrophosphorylase (AGPase), the rate limiting step of starch biosynthesis. Transgenic rice event (NR16+) with increased leaf AGPase activity (due to overexpression of the AGPase large and small subunit transgenes Sh2r6hs and Bt2) was subjected to RNA-sequencing. The results indicated that the leaf specific AGP transgene, which increased leaf starch, also had upregulation of the WRKY76 TF. Another regulatory protein, F-Box, was chosen as a candidate due to the F-box family's involvement in plant development. The current study examines the potential of these gene products for increasing starch biosynthesis in leaves via leaf specific overexpression. Results indicate that overexpression of WRKY76 increases leaf starch an average of 39% at both the one month and anthesis growth stages in comparison to the Varietal Control Nipponbare. WRKY76 transgenic lines have enhanced phenotype with an improved harvest index due to biomass and immature panicles trending down by 4% and 21% respectively, while seed weight trended 12% higher. Events overexpressing WRKY76 were also found to upregulate important starch biosynthetic and carbon metabolism genes including AGPL1, AGPS2, SSIIIb, GBSII, Rbcs, PRK, and GS2 as well as leading to a general upregulation of leaf tissue carbohydrates. Events 1, 2, and 12 additionally had on average 13% increased photosynthetic rate at the one month growth stage. The findings of this study support WRKY76 as a dynamic regulator of the starch biosynthetic pathway conferring more efficient carbon assimilation leading to an increased harvest index.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 Modification of a starch biosynthetic enzyme : potential for increased seed yield in wheat (Triticum aestivum)(Montana State University - Bozeman, College of Agriculture, 2002) Meyer, Fletcher DamienItem Biosynthesis of bromegrass mosaic virus ribonucleic acid(Montana State University - Bozeman, College of Agriculture, 1967) Branson, Dean RussellItem The fatty acid elongase of Physaria fendleri increases hydroxy fatty acid accumulation in transgenic Camelina(Montana State University - Bozeman, College of Agriculture, 2013) Snapp, Anna Rose; Chairperson, Graduate Committee: Chaofu LuPlant oils containing hydroxy fatty acids (HFA) are desirable for a wide variety of applications including lubricants, plasticizers, surfactants, polyesters, paints, sealants, biodiesel, and more. Due to unfavorable agronomic attributes of natural accumulators such as castor and lesquerella, many efforts have been made to produce hydroxyl fatty acids in crop plants. The hydroxy fatty acid synthesis pathway has been extensively studied and key genes such as the castor fatty acid hydroxylase, RcFAH, have been discovered. However, insertion of the RcFAH gene into various Arabidopsis backgrounds under the control of seed specific promoters failed to result in high accumulation of the desired HFA products, highlighting a need for more research to uncover additional constraints and factors affecting the fluxes involved with the accumulation of these unusual fatty acids in seed oil. In this study I investigated the effect of co-expressing a fatty acid elongase gene, LfKCS3, from Lesquerella (Physaria) fendleri along with the castor hydroxylase gene, RcFAH, on accumulation of hydroxyl fatty acids in seed oil of the crop plant Camelina sativa. On its own, wild type camelina contains no hydroxy fatty acids but insertion of the RcFAH gene results in accumulation of around 15% HFA in transgenic camelina, however, addition of the LfKCS3 gene resulted in a significant increase in very long chain 20-carbon hydroxyl fatty acids from <2% to 8%; total hydroxyl fatty acids also increased from 15% to 22% in the highest accumulating lines. The presence of the LfKCS3 enzyme effectively increased total HFA levels at all stages of oil accumulation in developing seeds while also decreasing the amount of these fatty acids left on the phospholipid, phosphatidylcholine. This combination of increased 20- carbon and total hydroxyl fatty acid accumulation along with the decreased HFA levels in phosphatidylcholine indicates that the LfKCS3 gene helps to enhance the flux of HFA out of phosphatidylcholine for incorporation of HFA into triacylglycerol, aiding in relief of the metabolic bottleneck for engineering economically viable levels of these fatty acids in oilseed crops.Item SrbA-regulation of ergosterol biosynthesis in Aspergillus fumigatus : gateway to azole resistance & hypoxia adaptation(Montana State University - Bozeman, College of Letters & Science, 2013) Blosser, Sara Jean; Chairperson, Graduate Committee: Robert Cramer; Robert A. Cramer was a co-author of the article, 'Srebp-dependent triazole susceptibility in Aspergillus fumigatus is mediated through direct transcriptional regulation of erg11A (cyp51A)' in the journal 'Antimicrobial agents and chemotherapy' which is contained within this thesis.; Brittney Hendrickson, Nora Grahl, Bridget M. Barker and Robert A. Cramer were co-authors of the article, 'Two C4-sterol methyl oxidases (erg25) catalyze ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus' submitted to the journal 'Molecular microbiology' which is contained within this thesis.; Robert A. Cramer was a co-author of the article, 'Removal of C4-methyl sterol accumulation in an Srebp-null mutant of Aspergillus fumigatus restores hypoxia growth' submitted to the journal 'PLoS pathogens' which is contained within this thesis.Aspergillus fumigatus is a human fungal pathogen and the primary cause of Invasive Aspergillosis (IA). A rise in susceptible patient populations has dramatically increased the incidence of IA, and led to the emergence of triazole antifungal drug resistance. Triazoles target Erg11, an enzyme involved in ergosterol biosynthesis. Ergosterol biosynthesis has been widely targeted for antifungal drug development, but little is known about this pathway in A. fumigatus. We have identified a transcription factor, SrbA, which mediates triazole susceptibility, growth in hypoxia and low iron, and virulence during IA. Transcriptional studies identify ergosterol biosynthesis as one of the major genetic targets of SrbA, including erg11 and erg25. In this study, we examined the mechanism of Delta srbA triazole susceptibility. Construction of an erg11A conditional expression strain in the Delta srbA background restored erg11A transcript levels and, consequently, wild-type sensitivity to fluconazole and voriconazole. However, pniiAerg11A-Delta srbA did not restore hypoxia growth or the total ergosterol defect of Delta srbA. Increased accumulation of C4-methyl sterols indicates that the Erg25-step of ergosterol biosynthesis is defective in these strains. A. fumigatus encodes for two C4-demethylases, erg25A and erg25B. Erg25A serves in a primary role over Erg25B, as Delta erg25A accumulates more C4-methyl sterol intermediates than Delta erg25B. That both erg25 genes retain function, and are not limited to a singular substrate is unique in the eukaryotic kingdom. Genetic deletion of both erg25 genes is lethal, and single deletion of these genes revealed alterations in ergosterol biosynthesis. Delta erg25A displayed moderate sensitivity to hypoxia, reactive oxygen species (ROS), and dithiothreitol, but was not required for virulence in a murine model of IA. Erg25 assists in the ability of A. fumigatus to grow in hypoxia, as construction of a strain that constitutively expresses erg25A in the Delta srbA background restored the hypoxia growth defect of Delta srbA. This restoration revealed substantial insufficiencies in pflavA-erg25A-Delta srbA when adapting to hypoxia, as this strain was hypersensitive to cell wall perturbation and ROS. Additionally, restoration of erg25A impacted triazole antifungal susceptibility of Delta srbA, demonstrating a complex feedback system involved in ergosterol biosynthesis. These results demonstrate SrbA's involvement in a dynamic stress adaptation program mediated in part through ergosterol biosynthesis.