For systems biology, it is important to describe the kinetic and thermodynamic properties of enzyme-catalyzed reactions and reaction cascades quantitatively under conditions prevailing in the cytoplasm. While in part I kinetic models based on irreversible thermodynamics were tested, here in part II, the influence of the presumably most important cytosolic factors was investigated using two glycolytic reactions (i.e., the phosphoglucose isomerase reaction (PGI) with a uni-uni-mechanism and the enolase reaction with an uni-bi-mechanism) as examples. Crowding by macromolecules was simulated using polyethylene glycol (PEG) and bovine serum albumin (BSA). The reactions were monitored calorimetrically and the equilibrium concentrations were evaluated using the equation of state ePC-SAFT. The pH and the crowding agents had the greatest influence on the reaction enthalpy change. Two kinetic models based on irreversible thermodynamics (i.e., single parameter flux-force and two-parameter Noor model) were applied to investigate the influence of cytosolic conditions. The flux-force model describes the influence of cytosolic conditions on reaction kinetics best. Concentrations of magnesium ions and crowding agents had the greatest influence, while temperature and pH-value had a medium influence on the kinetic parameters. With this contribution, we show that the interplay of thermodynamic modeling and calorimetric process monitoring allows a fast and reliable quantification of the influence of cytosolic conditions on kinetic and thermodynamic parameters.

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<p>Cytochrome P450s are a super-family of heme containing proteins that are found in all domains of life and are involved in the synthesis and breakdown of steroids, xenobiotics, and pharmaceuticals. Using five heterologously expressed zebrafish (Danio rerio) CYP1s, an assay was developed for CYP activity in order to monitor the consumption of the cofactor NADPH, providing a label-free screening tool to determine function of novel CYP genes. Using well-established fluorogenic substrates, NADPH and NADP+ were separated by capillary electrophoresis (CE) from stopped CYP1 reactions and measured with UV absorbance detection as a surrogate to assess the rate of substrate metabolism. Product formation was confirmed by fluorometric detection of metabolites, giving rates of enzyme activity which could be compared to the rates of cofactor turn-over measured by CE. 17β-estradiol, four alkoxyresorufin and two coumarin based synthetic fluorogenic CYP substrates were screened for activity with recombinant zebrafish CYP1A, 1B1, 1C2, 1C2 and 1D1. Cofactor consumption was generally much larger than product formation for the majority of substrates and CYP1 isoforms, suggesting that the majority of metabolic events were uncoupled. Large uncoupling was seen in CYP1 when metabolizing estradiol, showing that endogenous compounds can also show severe uncoupling. Reactive oxygen species (ROS), a product of uncoupled events, were detected with 2,7- dichorofluorescein. Attempts for concomitant detection of ROS production and cofactor consumption with CE-UV detection were investigated, however, detection limits for 2,7-dichlorofluorescein were not adequate for detection of hydrogen peroxide production from CYP1 mediated reactions. Future work will be required to develop a single assay to quantitatively measure CYP activity by CE for functional determination of CYPs with unknown function.</p> / Master of Science (MSc)

<p>The rise of bacterial infections and increase of antibiotic resistant bacteria has become a major problem in the treatment of bacterial infections. The use and overuse of antibiotics, and the inherent ability of bacteria to adapt to their environment, have lead to the emergence of strains that are resistant to all antibiotics. Ideally, new targets for antibacterial drug therapy would be essential to the virulence of most or all bacteria. That is, antibiotics exploiting these targets would have broad spectrum activity. 3-Deoxy-D-arabinoheptulosonate-7- phosphate (DAHP) synthase could be such a target. This enzyme catalyzes the condensation of erythrose 4-phosphate (E4P) and phoshoenolpyruvate (PEP) to form DAHP. The DAHP synthase-catalyzed reaction is the first committed step in the shikimic acid biosynthetic pathway leading to the aromatic amino acids and other secondary metabolites in all bacteria and some parasites. Inhibition of this enzyme would lead to a depletion of aromatic amino acids within the cell, halting new protein synthesis and killing the cells. Our lab has developed a transition state analogue, DAHP oxime, which is a slow binding, potent inhibitor of DAHP synthase. Kinetic characterization of inhibitor binding revealed DAHP oxime to be a competitive inhibitor with an ultimate Ki* of 81 nM. Crystal structures of DAHP oxime bound to DAHP synthase revealed that the inhibitor bound to two of the four subunits. The two unbound subunits remain catalytically competent, suggesting that DAHP synthase may utilize a half-of-sites mechanism during catalysis. We further investigated changes in DAHP synthase dynamics in response to PEP and DAHP oxime binding via solvent hydrogen/deuterium exchange mass spectrometry. DAHP synthase in the unbound form was loosely structured around the surface exposed regions, whereas the X-ray crystal structures appeared to be more fully structured. Binding of both PEP and DAHP oxime introduced different degrees of dynamic stabilization.</p> / Doctor of Philosophy (PhD)

<p>The food-borne pathogen, <em>Listeria monocytogenes</em>, is a global health concern as it has been responsible for multiple food contamination outbreaks over the past century. Current detection methods like the enzyme-linked immunoassays (ELISA), and polymerase chain reaction (PCR) take over 24 h to attain results, are costly, require specialized equipment and trained personnel. In this study we investigated the use of functional nucleic acid (FNA) to develop a rapid and cost-effective detection method for <em>L. monocytogenes</em>. We carried out in<em> vitro</em> selection in order to isolate a fluorescently labeled DNA-RNA hybrid strand that can be bound and cleaved by specific endoribonucleases (RNase) from <em>L. monocytogenes</em>. We termed these DNA-RNA hybrid strands RNase-cleaved fluorescent substrate (RFS). Since no past studies have isolated RNases from <em>L. monocytogenes</em>, we first identified the genes based on sequence similarities with well characterized RNases. We purified and characterized RNase HII, RNase III and RNase G. Since this study focused primarily on developing RFS for RNase HII, we performed an in depth <em>in vitro</em> biochemical analysis to characterize this enzyme. We found that RNase HII from <em>L. monocytogenes</em> plays an important role in DNA replication and repair. Furthermore, we obtained six sequence classes by <em>in vitro</em> selection which could interact with RNase HII. The key nucleotide regions involved with RNase HII interactions were identified. In the final study, we showed the sequences isolated by <em>in vitro</em> selection could also be used as a tool to study ribonuclease function and identify new interaction between enzyme and substrate.</p> / Master of Science (MSc)

Complex biosynthetic pathways are required for the assembly and insertion of iron-sulfur (Fe-S) cluster cofactors. The four cluster biogenesis systems that have been discovered require at least one ATPase, but generally the function of nucleotide hydrolysis is understudied. In the cytosolic iron sulfur cluster assembly (CIA) system, responsible for delivering [Fe4-S4] cluster cofactors for cytosolic and nuclear enzymes, the assembly scaffold comprises two homologous ATPases, called Nbp35 and Cfd1 in Saccharomyces cerevisiae. Genetic studies have discovered that the ATPase sites are required for scaffold function in vivo, but in vitro studies have failed to reveal why. The ATPase sites of the Nbp35 and Cfd1 contain a conserved P-loop nucleotide-binding protein fold with a deviant Walker A motif. Known metal trafficking P-loop NTPases’ metallochaperone mechanisms rely on both nucleotide binding and hydrolysis to properly assemble and deliver metal cargo. Furthermore, P-loop NTPases with a deviant Walker A motif commonly serve as central regulatory switches whose hydrolysis activity is modulated by small molecule cargos and/or protein partners. Therefore, it is proposed that the role of Nbp35-Cfd1’s ATPase sites is to direct Fe-S cluster movement by regulating protein and metal cargo interactions. The goal of this thesis is to better understand the scaffold reaction cycle by investigating the metallochaperone mechanism through Nbp35-Cfd1’s protein communications with its ATPase sites. To do this, the identification of at least one nucleotide-dependent partner protein must first be discovered. Herein, in vitro methods have been developed to uncover the scaffold’s ATPase site regulation of protein interactions. We describe a qualitative affinity copurification assay and a quantitative analysis for evaluating the dissociation constant and the kcat and Km values for ATP hydrolysis for the scaffold–partner protein complex. Additionally, the execution of these ATPase assays in an anaerobic environment can be applied to study nucleotide hydrolases involved in metallocluster biogenesis. These in vitro methods are applied to Nbp35-Cfd1 and it is discovered that ATP binding and hydrolysis regulates Nbp35-Cfd1 binding with two CIA factors: Dre2, a reductase proposed to assist in Fe-S cluster assembly, and Nar1, an adaptor between the early and late CIA factors. Although reconstitution of the scaffold’s Fe-S clusters results in a two-fold increase in its ATPase activity, the Dre2 and Nar1 ATP hydrolysis stimulation is dampened, demonstrating that both the Fe-S cargo and partner proteins regulate the scaffold’s ATPase reaction cycle. Next, the domains required for binding and ATPase stimulation were identified for Nbp35-Cfd1 with its partner proteins Dre2 and Nar1. The C-terminal Fe-S binding domain of Dre2 is sufficient for ATPase stimulation, while the Nar1 requires both its N- and C-terminal Fe-S binding domains to activate Nbp35-Cfd1’s ATP hydrolysis. The N-terminal Fe-S binding domain of Nbp35 is dispensable for binding and ATPase stimulation of both Dre2 and Nar1. The CIA targeting complex protein Cia1, which binds to Nar1, competes off Nbp35-Cfd1, indicating a shared binding domain. This data both validates and refines the current working model of the CIA system. To test whether the communication between the ATPase and Fe-S cluster binding domains of the CIA scaffold functions in an analogous manner across multiple species, a preliminary analysis was completed for whether Chaetomium thermophilum and Homo sapien Nbp35-Cfd1 exhibit similar ATPase characteristics and partner protein interaction as their S. cerevisiae ortholog. Human and fungal Nbp35-Cfd1 exhibit ATP binding and demonstrate nucleotide-dependent interactions with Dre2 and Nar1, suggesting that these interactions in a similar manner to effectively communicate in the CIA pathway. Overall, our study uncovers striking similarities between the CIA pathway and other systems which exploit a deviant Walker A NTPase to coordinate complex, multiprotein processes. Identification of the scaffold’s partner proteins significantly advances our understanding as to why the Nbp35/MRP-type Fe-S cluster biogenesis proteins are nucleotide hydrolases. This work provides some mechanistic insight into the functions of these proteins and provides a roadmap for how to investigate this large and widely distributed family and other P-loop NTPase metallochaperones. / 2024-09-19T00:00:00Z