The production of new drugs as potential pharmaceutical targets is arguably one of the most important avenues of medicine, as existing diseases not only require treatment, but it is also certain that new diseases will appear in the future which will need treatment. Indeed, existing medicines such as antibiotics and immunosuppressants maintain their current activities in their respective realms, yet the molecular and stereochemical complexity of these compounds cause a burden on organic synthetic chemists that may prohibit the high yields required to manufacture a drug. The enzyme systems that naturally manufacture these compounds are incredibly efficient in doing so, and also do not use environmentally harmful solvents, chiral auxiliaries, or metals that are utilized in the current syntheses of these compounds; therefore utilizing these enzymes' machinery for the biocatalysis of new medicinally-relevant compounds, as researchers have in the past, is undeniably a rewarding endeavor. In order to harness these systems' biocatalytic potential, we must understand the processes which they operate. This work focuses on ketoreductase domains, since they are responsible for setting most of the stereocenters found within these complex secondary metabolites. We have supplied a library of substrates to multiple ketoreductases to test their limits of stereospecificity and found that, for the most part, they maintain their natural product stereospecificity seen in nature. We were even able to convert a previously nonstereospecific ketoreductase to a stereospecific catalyst. We have also developed a new technique to follow ketoreductase catalysis in real-time, which can also differentiate between which diastereomeric product is being produced. Finally, we have elucidated the structure of a ketoreductase that reduces non-canonically at the [alpha]- and [beta]- position, and functionally characterized its activities on shortened substrate analogs. With the knowledge gained from this dissertation we hope that the use of ketoreductases as biocatalysts in the biosynthesis of new natural product-based medicines is a much nearer reality than before. / text

Altered cytoskeletal regulation impacts numerous physiological phenomena: cell motility, apoptosis, oncogenic transformation and parasitic infection. The protein tyrosine phosphatase (PTP)-PEST contains multiple motifs mediating its recruitment to signalling components, and is required for actin filament organization. However, little is known regarding either the importance of PTP-PEST subcellular localization, or the role of PTP-PEST in either parasitic infection or apoptosis. My doctoral research was therefore focussed on elucidating the effect of subcellular distribution on PTP-PEST activity, specifically with respect to regulation of p130Cas (a PTP-PEST substrate), as well as on the involvement of PTP-PEST in both host-pathogen relations and apoptosis. First, PTP-PEST was found both within the cytosol and at the plasma membrane. Using PTP-PEST -/- rescued cell lines, I observed that tyrosine phosphorylation-dependent p130Cas interactions were controlled primarily by cytosolic PTP-PEST. Secondly, infection of fibroblasts with Leishmania major was observed to induce dramatic actin rearrangements, and to alter the phosphorylation state of numerous proteins. Importantly, both PTP-PEST and p130Cas were processed by the parasitic protease GP63 during infection. GP63 was also required for the cleavage of additional host proteins: cortactin, TC-PTP and caspase-3. Of note, Leishmania parasites mediated p38 inactivation, correlating with the proteolysis of its upstream activator TAB1, in a GP63dependent manner. These results indicate that GP63 plays a key role in a number of biochemical events, potentially contributing to Leishmania infectivity. Finally, PTP-PEST was found to relocalize to the edges of retracting membrane ruffles of apoptotic cells. Surprisingly, PTP-PEST was specifically cleaved by caspase-3 at the 549DSPD motif during apoptosis; leading to modification of catalytic activity and scaffolding properties, and sensitizing cells to Fas-mediated detachment. As this data demonstrated a potential role for caspase cleavage in PTP regulation, I also investigated the presence of conserved putative caspase-c1eavage sites in other family members. In summary, the data presented herein links PTP-PEST with various biological processes: oncogenic signalling, host-pathogen interactions, and apoptosis. In addition to demonstrating the involvement of PTP-PEST in diverse signalling pathways, these studies underscore the importance of subcellular localization and proteolysis in the regulation of this PTP.

This thesis presents evidence for a novel mechanism of regulation of pyruvate dehydrogenase (PDH) kinase by fatty acids and also results of a study of muscle triacylglycerol concentration. In animals regulation of PDH complex activity is central to the selection of respiratory fuels and to the conservation of glucose during carbohydrate deprivation. The principal means of regulation of PDH complex is interconversion of phosphorylated (inactive) and dephosphorylated (active) forms effected by PDH kinase and PDH phosphatase. Earlier in vitro studies by others had identified both shorter term (min) and longer term (hours) mechanisms of activation of PDH kinase by fatty acid. In the present study PDH kinase activity (as measured by rates of ATP-dependent inactivation of PDH complex in extracts) was shown to be increased when rat heart mitochondria were incubated with palmitoyl-L-carnitine [PC] (and other CoA utilising respiratory substrates). The activation of PDH kinase persisted through removal of respiratory substrate following incubation with CCCP. A comparable effect of PC was also demonstrable in heart mitochondria from 48h-starved rats (i.e. the mechanism may be distinct from that which increases PDH kinase activity in starvation). Rates of ATP-dependent inactivation of PDH complex were also increased when extracts of rat heart mitochondria were incubated with palmitoyl-CoA (PCoA); the increase was comparable with that seen on incubation of intact mitochondria with PC. The PC effect in intact mitochondria and the PCoA effect in mitochondrial extracts may not be identical as PCoA further increased PDH kinase activity in extracts from mitochondria incubated with PC. Rates of incorporation of ³²P from [γ-<sup>32</supP]ATP into PDH complex were unaltered by pnor incubation of mitochondria with PC or by pnor incubation of mitochondrial extracts with PCoA. Three lines of evidence confirmed that the effect of PC to accelerate ATP-dependent inactivation involved phosphorylation of the PDH complex (viz; use of a non-phosphorylatmg ATP analogue; use of known inhibitors of PDH kinase; and use of known activators/inhibitors of PDH phosphatase). Earlier studies had shown that phosphorylation in punfied bovine and porcine PDH complexes is half site (involves only one α-chain in E1 (α2β2) and had suggested that phosphorylation in rat heart complex may be full site (i.e. involves both α-chains). The present study suggests the possibility that elevation of fatty acyl CoA under slaughter house conditions might be a determinant of half site phosphorylation. A method was developed and evaluated for measurement of triacylglycerol in rat soleus muscle strips with the object of investigating factors that may regulate triacylglycerol synthesis in this muscle. This study was abandoned because, although the method was highly reproducible, great variation was found in the triacylglycerol concentration of individual muscles suggesting the possibility of variable contamination with small amounts of adipose tissue.

Thesis (Ph. D.)--University of Wyoming, 2006. / Title from PDF title page (viewed on Nov. 15, 2007). Includes bibliographical references.

Thesis (M.S. in Soil Science)--Washington State University. / Includes bibliographical references.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Dr. Suzanne B. Shuker, Committee Chair ; Dr. Thomas M. Orlando, Committee Co-Chair ; Dr. Donald F. Doyle, Committee Member ; Dr. C. David Sherrill, Committee Member ; Dr. Andreas S. Bommarius, Committee Member ; Dr. S. Michele Owen, Committee Member ; Dr. Vicky L. H. Bevilacqua, Committee Member.

abstract: DNA has recently emerged as an extremely promising material to organize molecules on nanoscale. The reliability of base recognition, self-assembling behavior, and attractive structural properties of DNA are of unparalleled value in systems of this size. DNA scaffolds have already been used to organize a variety of molecules including nanoparticles and proteins. New protein-DNA bio-conjugation chemistries make it possible to precisely position proteins and other biomolecules on underlying DNA scaffolds, generating multi-biomolecule pathways with the ability to modulate inter-molecular interactions and the local environment. This dissertation focuses on studying the application of using DNA nanostructure to direct the self-assembly of other biomolecular networks to translate biochemical pathways to non-cellular environments. Presented here are a series of studies toward this application. First, a novel strategy utilized DNA origami as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multi-component systems from biological scaffolds using the power of rationally engineered DNA nanostructures. Next, discrete glucose oxidase (GOx)/ horseradish peroxidase (HRP) enzyme pairs were organized on DNA origami tiles with controlled interenzyme spacing and position. This study revealed two different distance-dependent kinetic processes associated with the assembled enzyme pairs. Finally, a tweezer-like DNA nanodevice was designed and constructed to actuate the activity of an enzyme/cofactor pair. Using this approach, several cycles of externally controlled enzyme inhibition and activation were successfully demonstrated. This principle of responsive enzyme nanodevices may be used to regulate other types of enzymes and to introduce feedback or feed-forward control loops. / Dissertation/Thesis / Ph.D. Biochemistry 2013

Foram analisados alguns parâmetros que influenciam os bioprocessos, submerso e sólido, do fungo Eupenicillium javanicum na produção de peptidases. No bioprocesso submerso foram avaliados a influência de diferentes concentrações e tipos de fonte de carbono e concentrações de fonte nitrogênio no meio, pH, temperatura e tempo de incubação. No bioprocesso sólido avaliou-se a influência de dois tipos de resíduos agroindustriais, em diferentes proporções, dois tipos de fonte de nitrogênio, em diferentes concentrações, tempo e temperatura de incubação. As peptidases produzidas em ambos os bioprocessos foram caracterizadas bioquimicamente, avaliando pH e temperatura ótima, estabilidade em diferentes temperaturas e valores de pH e influência da adição de íons e inibidores na atividade da peptidase. A enzima produzida em bioprocesso sólido foi submetida ao processo de purificação utilizando métodos cromatográficos. Utilizando a peptidase pura realizou-se a caracterização bioquímica funcional e a determinação dos parâmetros cinéticos, ambos utilizando o substrato peptídico de supressão intramolecular de fluorescência. Além disso, o extrato enzimático obtido através do bioprocesso foi submetido ao processo de microencapsulação, visando uma maior estabilidade das peptidases e facilidade no armazenamento e transporte, utilizando a técnica de Spray drying, em seguida foi avaliado o rendimento do processo e a estabilidade das peptidases das micropartículas produzidas. O fungo Eupenicillium javanicum mostrou potencial na produção de peptidases em ambos bioprocessos, produzindo peptidases da classe das metalopeptidases com alta estabilidade em diferentes valores de pH e temperatura. O processo de purificação mostrou-se viável e reprodutível. A análise dos parâmetros cinéticos revelou uma grande influência do lado \"linha\" da peptidase na eficiência catalítica. O processo de microencapsulação mostrou-se viável e gerou micropartículas estáveis. A peptidase produzida apresentou características que demonstram seu potencial uso nas diferentes áreas industriais. / Some parameters that influence the submerged and semi solid bioprocesses, by the fungus Eupenicillium javanicum in the peptidases production, were conducted. In submerged bioprocess was evaluated the effect of different concentrations and types of carbon source and nitrogen source in the medium, pH, temperature and incubation time. In semi solid bioprocess semi solid was evaluated the influence of two types of agroindustrial residues, in different proportions, and different concentrations of nitrogen source, temperature and of incubation time. The peptidases produced in both bioprocesses were characterized biochemically, evaluating, the optimum pH and temperature, stability at different temperatures and pH values and the influence of the addition of ions and inhibitors on peptidase activity. The enzyme produced in semi solid bioprocess was subjected to the purification process using chromatographic methods. Using pure peptidase was performed the biochemical characterization and determination of kinetic parameters, both using the fluorescence intramolecular suppression peptide substrate. Furthermore, the enzymatic extract obtained by semi solid bioprocess was subjected to microencapsulation process by using the technique of spray drying, in order to obtain greater stability, ease storage and transport of peptidases, then assessed the yield process and stability of the peptidases of microparticles produced. The fungus Eupenicillium javanicum showed potential production of peptidases in the both bioprocesses, producing peptidases that belong to the class of metallopeptidases, with high stability at different pH and temperature. The purification process was feasible and reproducible. The analysis of kinetic parameters revealed a strong influence of the \"line\" side on the peptidase catalytic efficiency. The microencapsulation process was feasible and generated stable microparticles. The peptidase produced has characteristics that demonstrated it a potential use in different industrial fields.

No description available.

Abstract Fatty acid degradation in living organisms occurs mainly via the β-oxidation pathway. When this work was started, it was known that the hydration and dehydrogenation reactions in mammalian peroxisomal β-oxidation were catalyzed by only multifunctional enzyme type 1 (MFE-1; Δ2-Δ3-enoyl-CoA isomerase/2-enoyl-CoA hydratase 1/(3S)-hydroxyacyl-CoA dehydrogenase) via the S-specific pathway, whereas in the yeast peroxisomes via the R-specific pathway by multifunctional enzyme type 2 (MFE-2; 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl-CoA dehydrogenase). The work started with the molecular cloning of the rat 2-enoy-CoA hydratase 2 (hydratase 2). The isolated cDNA (2205 bp) encodes a polypeptide with a predicted molecular mass of 79.3 kDa, which contains a potential peroxisomal targeting signal (AKL) in the carboxyl terminus. The hydratase 2 is an integral part of the cloned polypeptide, which is assigned to be a novel mammalian peroxisomal MFE-2. The physiological role of the mammalian hydratase 2 was investigated with the recombinant hydratase 2 domain derived from rat MFE-2. The protein hydrates a physiological intermediate (24E)-3α, 7α, 12α-trihydroxy-5β-cholest-24-enoyl-CoA to (24R, 25R)-3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl-CoA in bile acid synthesis. The sequence alignment of human MFE-2 with MFE-2(s) of different species reveals 12 conserved protic amino acid residues, which are potential candidates for catalysis of the hydratase 2. Each of these residues was replaced by alanine. Complementation of Saccharomyces cerevisiae fox-2 (devoid of endogenous MFE-2) with human MFE-2 provided a model system for examing the in vivo function of the variants. Two protic residues, Glu366 and Asp510, of the hydratase 2 domain of human MFE-2 have been identified and are proposed to act as a base and an acid in catalysis. Mammalian MFE-2 has a (3R)-hydroxyacyl-CoA dehydrogenase domain, whereas the yeast MFE-2 has two dehydrogenase domains, A and B. The present work, applying site-directed mutagenesis to dissect the two domains, shows that the growth rates of fox-2 cells expressing a single functional domain are lower than those of cells expressing S. cerevisiae MFE-2. Kinetic experiments with the purified proteins demonstrate that domain A is more active than domain B in catalysis of medium- and long-chain (3R)-hydroxyacyl-CoA, whereas domain B is solely responsible for metabolism of short-chain substrates. Both domains are required when yeast cells utilize fatty acids as the carbon source.