The Fdc1 and Pad1 decarboxylase system from Saccharomyces cerevisiae has been identified as a potential candidate to feature in novel biofuel production pathways based on its ability to catalyze the transformation of sorbic acid, an antimicrobial compound, to 1, 3-pentadiene, a volatile hydrocarbon. Although information on the system is currently insufficient to permit a full assessment of its potential for future commercialization, it is hoped that (rational) engineering approaches can be used to evolve the enzymes to produce more desirable hydrocarbons. This requires biochemical characterization of the proteins. Genetic manipulation experiments have indicated that both enzymes are required for activity. However, no in vitro studies were conducted to verify the function, determine the relationship or establish the cofactor requirements of Fdc1 and Pad1. Results reported here establish that Fdc1 is the enzyme responsible for catalyzing decarboxylation, requiring a novel cofactor synthesized by Pad1 (or the bacterial homologue UbiX) for activity. High resolution crystal structures and mass spectrometry data from Fdc1 co-expressed with UbiX have indicated that the cofactor corresponds to a modified flavin mononucleotide (FMN) that has been extended with a C5-unit through linkages at the N5 and C6 atoms, creating a fourth, non-aromatic ring on the isoalloxazine group. Subsequent solution studies have established that this modification is achieved through isoprene chemistry, with UbiX facilitating prenyl transfer from dimethylallyl monophosphate (DMAP) to FMN. Analysis of wild type and mutant UbiX constructs by kinetic X-ray crystallography has allowed several distinct stages of the prenyl transfer reaction to be trapped, establishing that the protein uses a number of chemical strategies similar to terpene synthases to generate its product. The active site is dominated by pi systems, which aid heterolytic cleavage of the isoprene precursors phosphate-C1’ bond following FMN reduction, leading to the formation of an N5-C1’ intermediate. UbiX then acts as a chaperone for adduct reorientation, potentially via a transient tertiary carbocation, ultimately resulting in ring closure between the C6 and C3’. This work has established the biochemical principles underpinning the Fdc1 and Pad/UbiX decarboxylase system, providing a platform from which rational evolution approaches can be applied to the enzymes, specifically Fdc1, to improve their validity in the biofuels industry. It has also identified a novel cofactor that extends the previously well-documented flavin and isoprenoid repertoire.

Reclaimed roads on the Little Missouri National Grasslands of southwestern North Dakota have not returned to pre-disturbance conditions. Phospholipid fatty acid analysis was performed on soil samples collected from reclaimed roads and adjacent prairie to assess reclamation effects on the microbial community. Additionally, nutrient cycling capacity was measured by four enzyme assays. Ordination analysis of PLFA data identified a distance gradient indicating microbial communities of reclaimed roads were different from the prairie. Specifically, Gram-negative bacteria and arbuscular mycorrhizal fungi are associated with roads; soil organic matter was associated with prairie sites. Soil enzyme activities associated with prairie sites indicate greater nutrient cycling. The soils of reclaimed roads have not accumulated sufficient organic matter to sustain both plant and microbial communities characteristic of the surrounding prairie.

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Our laboratory is interested in studying the junction of glycolysis and the tricarboxylic acid (TCA) cycle, specifically the enzymes phosphoenolpyruvate carboxykinase, pyruvate carboxylase and phosphoenolpyruvate carboxylase. All produce oxaloacetate (OAA) for the cell. OAA production is critical for cell carbon synthesis in the methanogenic archaea. Therefore OAA-generating enzymes are essential for the survival of methanogens. In part of this study we investigated archaeal-type phosphoenolpyruvate carboxylase (PpcA), a new type of phosphoenolpyruvate carboxylase, which is widespread in the archaea and is found in three bacterial species. The form of phosphoenolpyruvate carboxylase (Ppc) that is prevalent in bacteria and plants is not found in the archaea. Due to complications expressing PpcA in the soluble form and difficulty purifying this enzyme from methanogens, an in-depth investigation of this enzyme's biochemical properties has yet to occur. In this study we demonstrate the successful expression of a PpcA homolog in the soluble fraction of Escherichia coli. We purified the recombinant protein to homogeneity. This development provides the means to study the enzyme's biochemical properties and manipulate the primary sequence in order to identify residues critical to the enzyme's function. We also show that this PpcA homolog does have the postulated activity and investigate its biochemical properties. The data show that PpcA has unique properties in regard to the enzyme's substrate and its regulation by metabolites. Our data also reveal that PpcA is a membrane associated protein, unlike Ppc, which is a soluble protein. We also show that pyruvate carboxylase (Pyc) can be expressed recombinantly in Pseudomonas aeruginosa at levels sufficient for structure-function studies. This is a major step forward in the expression in Pyc because it cannot be expressed at high levels in Escherichia coli. These are important developments in studying the enzymes that connect glycolysis and the TCA cycle. / Master of Science

The mitochondrial membrane bound flavoenzymes monoamine oxidase A and B (MAO-A and MAO-B) catalyze the a-carbon oxidation of a variety of amines including neurotransmitters such as dopamine and serotonin. Although the primary structures of these enzymes have been established from the corresponding gene sequences, relatively little is known regarding the structural features of the active sites which lead to the selectivities observed with various substrates and inhibitors. In spite of many efforts, these enzymes have not been crystallized. In the absence of X-ray structures, the design, synthesis, and evaluation of biological activity remain the only way to assess a view of the active sites, through SAR and QSAR studies. The excellent MAO-A and/or B substrate and inhibitor properties of various 1,4-disubstituted-1,2,3,6-tetrahydropyridine derivatives offer an interesting opportunity to probe the active sites of MAO-A and MAO-B. In an effort to explore the spatial features of the active sites, we have synthesized series of substituted tetrahydropyridines, evaluated their biological activity with purified MAO-A and MAO-B, and carried out a topological analysis of the MAO active sites using molecular modeling. In addition, the results described in this thesis provide evidence that the MAO-A and MAO-B active sites differ in shape, regions of activity, and areas that tolerate polar interactions. The role of MAO in neurodegenerative processes such as Parkinson's Disease has been recognized for some time. The structurally unique parkinsonian inducing substrate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is bioactivated to neurotoxic metabolites. The mechanism of neurotoxicity has been studied extensively and it is known that MAO-B catalyzes the conversion of MPTP to the 2,3-dihydro-1-methyl-4-phenylpyridinium species (MPDP+) which undergoes further oxidation to the neurotoxic metabolite 1-methyl-4-phenyl pyridnium (MPP+). However, the role of MAO-A in mediating a neurotoxic response, has not been fully defined due to the lack of selective MAO-A substrates. In this thesis, we have investigated the neurotoxic potential of several tetrahydropyridines in C57Bl/6 mice and the ability of selective inhibitors to protect against the expression of MAO mediated neurotoxicity. / Ph. D.

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 69-72). Also available on the World Wide Web.

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This study has determined the cellular site of adenosine deaminase (ADA) expression in the mouse during development from Days 5 through 13 (day vaginal plug was found = Day 0) of gestation. Developmental expression of ADA progressed in two overlapping phases defined genetically (maternal vs. embryonal) and according to region (decidual vs. placental). In the first phase, ADA enzyme activity increased almost 200-fold in the antimesometrial region (decidua capsularis + giant trophoblast cells) from Days 6 through 9 of gestation but remained low in the mesometrial region. Immunohistochemical staining revealed a major localization of ADA to the secondary decidua. In the second phase, ADA activity increased several-fold in the placenta (labyrinth + basal zones) from Days 9 through 13 of gestation but remained low in the embryo proper. Immunohistochemical staining revealed a major localization of ADA to secondary giant cells, spongiotrophoblast, and labyrinthine trophoblast. Regression of decidua capsularis and growth of the spongiotrophoblast population accounted for an antimesometrial to placental shift in both ADA enzyme activity and a 40-kDa immunoreactive protein band. To verify a shift from maternal to fetal expression, studies were performed with two strains of mice (ICR, Eday) homozygous for a different ADA isozyme (ADA-A, ADA-B). Blastocysts homozygous for Adab were transferred to the uterus of pseudopregnant female recipients homozygous for Adaa. The isozymic pattern in chimeric embryo-decidual units analyzed at Days 7, 9, 11, and 13 revealed a predominance of maternal-encoded enzyme at Days 7 through 11 of gestation and a shift to fetal-encoded enzyme by Day 13. Thus, maternal expression of ADA in the antimesometrial decidua may play a role during establishment of the embryo in the uterine environment, whereas fetal expression of ADA in the trophoblast might be important to placentation.

Thesis (PhD)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Pantothenate kinase (PanK) enzymes catalyze the first reaction in the five step biosynthesis of the essential cofactor coenzyme A. Enzymes representing each of the three identified PanK types have been studied and characterized and these PanK types exhibits a unique diversity between different organisms, therefore highlighting them as potential drug targets. In this study the type III PanK of specifically pathogenic bacteria were characterized with the goal of developing type-specific inhibitors. Several questions about the activity of the Mycobacterium tuberculosis enzyme was answered, which addresses the contradicting results achieved in related PanK studies performed to date. Furthermore the first inhibitors, that are competitive to the pantothenate binding site, were designed, synthesized and tested against the Pseudomonas aeruginosa enzyme. This resulted in the discovery of the most potent inhibitors of the type III PanKs to date. / AFRIKAANSE OPSOMMING: Pantoteensuurkinase-ensiem (PanK) kataliseer die eerste stap in die vyf stap biosintese van die lewens belangrike en essensiële kofaktor, koënsiem A (KoA). Die meerderheid patogeniese bakterieë, waaronder die organisme wat tuberkulose veroorsaak, besit ‘n unieke vorm van die PanK-ensiem. Gevolglik word hierdie ensieme as belangrike teikens vir die ontwikkeling van antibakteriële middels beskou. In hierdie studie is die aktiwiteit van die Mycobacterium tuberculosis ensiem gekarakteriseer wat verskeie teenstrydige bevindings oor hierdie ensiem beantwoord het. Verder is nuwe inhibitore vir die Pseudomonas aeruginosa ensiem ontwerp, gesintetiseer en getoets. Die beste inhibitore van hierdie tipe ensiem tot op hede is sodoende geïdentifiseer.