Orientador: Anete Pereira de Souza / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T21:21:36Z (GMT). No. of bitstreams: 1 Saraiva_AntonioMarcos_D.pdf: 12032714 bytes, checksum: 1262a05ca10735e855fa138a2093d04b (MD5) Previous issue date: 2009 / Resumo: A linhagem 9a5c da bactéria Xylella fastidiosa foi o primeiro fitopatógeno a ter seu genoma completamente sequenciado, o qual gerdu diversas informações sobre seu metabolismo e patogenicidade. Das orfs codificadas por esta bactéria, destaca-se; no presente trabalho, a XF0703, cuja proteína correlata (com 28,3 kDa) possui similaridade com proteínas SurE de várias outras bactérias. Proteínas SurEs são nucleotidases que desfosforilam diversos nucleosideos monofosforilados para seus respectivos nucleosideos. Tal função é de fundamental importância para manter o pool balanceado dos quatro (deoxi)ribonucleosideos para síntese de DNA e RNA, respectivamente. Este trabalho descreve a clonagem da orfXF0703 no vetor pET29a, a expressão da proteína recombinante (XfSurE) em Escheríchia coli BL21(DE3) e a purificação da mesma por cromatografia de afinidade ao níquel. A análise da estrutura secundária foi feita por dicroísmo circular e realizou-se a determinação do estado oligomérico por cromatografia de gel filtração e espalhamento de luz a baixo ângulo (SAXS), os quais revelaram que a proteína é um tetrâmero. Dados de caracterização funcional indicam que a proteína possui maior atividade em pH neutro na presença do íon manganês como cofator, com uma maior afinidade pelo substrato 3'-AMP (K0,5=0,16 mM). Além disso, ensaios cinéticos mostram que a proteína possui um comportamento alostérico com alta cooperatividade positiva (coeficiente de Hill em torno de 2,6) com todos os quatro substratos naturais testados (3'-AMP, 5'-dAMP, 5'-AMP e 5-GMP). Experimentos com a técnica de SAXS permitiram calcular o raio de giro (32,7 ± 0.2 A), distância máxima intramolecular (100 A) e a simetria do envelope da molécula (222). A estrutura de diversas SurEs homólogas já cristalizadas foram superpostas ao envelope obtido, sendo que StSurE (SurE de Salmonella com maior idenjidade de aminoácidos) mostrou ter o melhor ajuste. No entanto, notou-se que havia espaços vazios no envelope de XfSurE e tais espaços podiam ser preenchidos a partir do afastamento das alças responsáveis pela tetramerização e pela rotação dos f dímeros. Estes movimentos (translação e rotação) podem explicar o comportamento alostérico da proteína, facilitando a entrada de substrato ao sítio catalítico da molécula. / Abstract: The 9a5c strain from bacterium Xylella fastidiosa was the first phytopathogen to have its genome completely sequenced, which revealed a lot of information about its metabolism and its pathogenicity. 'From a variety of orfs encoded by this bacterium, we highlight, in this work, the XF0703, which correlated protein (with 28.3 kDa) has similarity with SurE proteins from several other bacteria. The SurE proteins *are nucleotidases that dephosphorylate various monophosphorylated nucleosides to their respective nucleosides. This function is critical for maintaining the balanced pool of four (deoxy) ribonucleosides for DNA and RNA synthesis. In this work, we describes the cloning of the XF0703 orf into the vector pET29a, the recombinant protein overexpression (XfSurE) in Escherichia coli BL21(DE3) and the protein purification by nickel affinity chromatography. The secondary structure analysis was done by circular dichroism, while oligomeric state determination was achieved by gel filtration chromatography and small-angle X-ray light scattering (SAXS), which showed that the protein is a tetramer. Functional characterization data indicate that the protein has a highest activity at neutral pH in the presence of manganese as a cofactor, with a highest affinity for the 3-AMP substrate (K0,5 = 0,16 mM). Furthermore, kinetic tests showed that the protein has a allosteric behavior with a high positive cooperativity (Hill coefficient around 2.6) for all natural substrates screened (3-AMP, 5'-dAMP, 5'-AMP and 5'-GMP). Experiments with SAXS technique have allowed to calculate the radius of gyration (32.7 ± 0.2 A), maximum intramolecular distance (100 A) and molecule symmetry. / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Stopped-flow experiments in which the NAD-malic enzyme was preincubated with different reactants at near saturating substrate concentrations suggest a slow isomerization of the E:NAD:Mg complex. The lag is eliminated by preincubation with Mg˙² and malate suggesting that the formation of E:Mg:Malate either bypasses or speeds up the slow isomerization step. Circular dichroic spectral studies of the secondary structural changes of the native enzyme in the presence and absence of substrates supports the existence of conformational changes with NAD˙ and malate. Thus, a slow conformational change of the E:NAD:Mg complex is likely one of the rate-limiting steps in the pre-steady state.

Plasmodium falciparum 1–deoxy–D–xylulose–5 phosphatereductoisomerase (PfDXR) plays a role in isoprenoid biosynthesis in the malaria parasite and is absent in the human host, making this parasite enzyme an attractive target for antimalarial drug design. To characterize PfDXR, it is necessary to produce large quantities of the enzyme in a soluble and functional form. However, the over–production of malarial proteins in prokaryotic host systems often results in the formation of truncated proteins or insoluble protein aggregates. A heterologous expression system was developed for the production of active PfDXR using codon harmonization and tight control of expression in the presence of lac repressor. Yields of up to 2 mg/l of enzyme were reported using the optimised expression system, which is 8 to 10– fold greater than previously reported yields. The kinetic parameters Km, Vmax and kcat were determined for PfDXR; values reported in this study were consistent with those reported in the literature for other DXR enzymes. A three–dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure–checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non–apicomplexan DXR proteins. Residues Val44 and Asn45, essential for NADPH binding; and catalytic hatch residues Lys224 and Lys226, which are unique to the species of Plasmodium, were mutated to resemble those of E. coli DXR. Interestingly,these mutations resulted in significant reductions in substrate affinity, when compared to the unmutated PfDXR. Mutant enzymes PfDXR(VN43,44AG) and PfDXR(KK224,226NS) also demonstrated a decreased ability to turnover substrate by 4–fold and 2–fold respectively. This study indicates a difference in the role of the catalytic hatch of PfDXR with regards to the way in which it captures substrates. The study also highlights subtle differences in cofactor binding to PfDXR, compared with the well characterized EcDXR enzyme. The validated PfDXR model was also used to develop a novel efficient in silico screening method for potential tool compounds for use in the rational design of novel DXR inhibitors. Following in silico screening of 46 potential DXR inhibitors, a two–tiered in vitro screening approach was undertaken. DXR inhibition was assessed for the 46 novel compounds using an NADPH– ependant DXP enzyme inhibition assay and antimalarial potential was assessed using P.falciparum–infected erythrocyte growth assays. Select compounds were tested in human cells in order to determine whether they were toxic to the host. From the parallel in silico and in vitro drug screening, it was evident that only a single compound demonstrated reasonable potential binding to DXR (determined using in silico docking), inhibited DXR in vitro and inhibited P. falciparum growth, without being toxic to human cells. Its potential as a lead compound in antimalarial drug development is therefore feasible. Two outcomes were evident from this work. Firstly, analogues of known antimalarial natural products can be screened against malaria, which may then lead towards the rational design of novel compounds that are effective against a specific antimalarial drug target enzyme, such as PfDXR. Secondly, the rational design of novel compounds against a specific antimalarial drug target enzyme can be untaken by adopting a coupled in silico and in vitro approach to drug discovery.

In studies of enzyme kinetics, reaction time courses are often condensed into a single set of initial rates describing the rate at the start of the reaction. This set is then analysed with the Henri-Michaelis-Menten equation. However, this process necessarily removes information from experimental data and diminishes its statistical significance due to a reduction of available data points. Further, if the examined system does not approach steady-state rapidly, the application of the steady-state-assumption can lead to flawed conclusions. Here, the analysis of two complex enzyme systems by numerical integration of kinetic rate equations is demonstrated. DNA polymerase catalyses the synthesis of DNA in a reaction that involves two substrates, DNA template and dNTP, both of which are highly heterogeneous in nature. The tight binding of DNA to DNA polymerase and its polymer properties prohibit the application of the initial-rate approach. By combining an explicit DNA binding step with a steady-state dNTP incorporation on a template of finite length, the DNA binding parameters and the dNTP incorporation steady-state parameters were estimated from processive polymerisation data in a global regression analysis. This approach is described in Chapter 2 and the results are in good agreement with previously published values. Further properties were investigated in studies of the temperature dependence and solvent isotope dependence of the kinetics. The processive polymerisation of DNA template was monitored using the fluorophore PicoGreen in a simple and inexpensive method described in Chapter 3. The catalytic cycle of ethanolamine ammonia lyase involves the homoloysis of the Co-C bond within the intrinsic B12 cofactor. This homolysis results in the formation of a Co(II)-adenosyl radical intermediate, which can be monitored using stopped-flow spectroscopy. The stopped-flow transients observed for EAL and related enzymes have long been difficult to analyse and interpret, possibly due to rapid methyl group rotation on the substrate. In Chapter 4 of this thesis we were able to rationalise this behaviour using numerical integration of the rate equations of a branched 16-state-kinetic model to fit stopped-flow transients in a global regression analysis. We were able to determine some intrinsic rate constants, and showed that the initial hydrogen atom transfer step is unlikely to have an inflated primary kinetic isotope effect, despite previous claims. More generally, this study demonstrates that the numerical integration analysis used here is likely to be applicable to a broad range of enzyme reaction kinetics.

Systems biology attempts to understand biological systems through the generation of predictive models that allow the behaviour of the system to be simulated in silico. Metabolic systems biology has in recent years focused upon the reconstruction and constraint-based analysis of genome-scale metabolic networks, which provide computational and mathematical representations of the known metabolic capabilities of a given organism. This thesis initially concerns itself with the development of such metabolic networks, first considering the community-driven development of consensus networks of the metabolic functions of Saccharomyces cerevisiae. This is followed by a consideration of automated approaches to network reconstruction that can be applied to facilitate what has, until recently, been an arduous manual process. The use of such large-scale networks in the generation of dynamic kinetic models is then considered. The development of such models is dependent upon the availability of experimentally determined parameters, from omics approaches such as transcriptomics, proteomics and metabolomics, and from kinetic assays. A discussion of the challenges faced with developing informatics infrastructure to support the acquisition, analysis and dissemination of quantitative proteomics and enzyme kinetics data follows, along with the introduction of novel software approaches to address these issues. The requirement for integrating experimental data with kinetic models is considered, along with approaches to construct, parameterise and simulate kinetic models from the network reconstructions and experimental data discussed previously. Finally, future requirements for metabolic systems biology informatics are considered, in the context of experimental data management, modelling infrastructure, and data integration required to bridge the gap between experimental and modelling approaches.

An approach to investigating cancer that has recently seen resurgence of interest is the “Warburg effect”. Otto Warburg originally described the altered metabolism of cancer cells and identified that they exhibit an increase in glucose uptake and lactate production. This up-regulation of glycolytic flux and glucose transport is now associated with 90% of cancers. In order to improve the overall understanding of the “Warburg effect” two forms of systems biology have been implemented - comparative in vitro analysis of kinetic activities and dynamic modelling. In this analysis, human breast cancer cell lines MCF-7, MDA-MB-231 and T47D and a non transformed breast cell line MCF-10A were used to identify key similarities and differences in kinetic activities across the glycolytic pathway. Additionally, activities of key glycolytic enzymes hexokinase, pyruvate kinase and lactate dehydrogenase were compared under hypoxic conditions to further understand regulation of cancer cells. The most prominent feature that arose from comparing the kinetic activities of the three malignant and one non-malignant cell line is that each cell line has its own specific set of activities for glycolysis. This indicates that there are differences in regulation across the glycolytic pathway for each of these cell lines. This is of specific interest in the search for therapeutic targets. Further, we determined that despite the prominence of oncogenic HIF signalling activities of hexokinase, pyruvate kinase and lactate dehydrogenase were further modulated by growth under hypoxic conditions. Despite the lack of obvious distinct kinetic differences between the non-cancerous and cancerous cells lines some discernible differences are apparent when modelled in silico.

The isotope partitioning studies of the Ascaris suum NAD-malic enzyme reaction were examined with five transitory complexes including E:NAD, E:NAD:Mg, E:malate, E:Mg:malate, and E:NAD:malate. Three productive complexes, E:NAD, E:NAD:Mg, and E:Mg:malate, were obtained, suggesting a steady-state random mechanism. Data for trapping with E:14C-NAD indicate a rapid equilibrium addition of Mg2+ prior to the addition of malate. Trapping with 14C-malate could only be obtained from the E:Mg2+:14C-malate complex, while no trapping from E:14C-malate was obtained under feasible experimental conditions. Most likely, E:malate is non-productive, as has been suggested from the kinetic analysis. The experiment with E:NAD:malate could not be carried out due to the turnover of trace amounts of malate dehydrogenase in the pulse solution. The equations for the isotope partitioning studies varying two substrates in the chase solution in an ordered terreactant reaction were derived, allowing a determination of the relative rates of substrate dissociation to the catalytic reaction for each of the productive transitory complexes. NAD and malate are released from the central complex at an identical rate, equal to the catalytic rate.

<p>HMG-CoA reductase from Pseudomonas mevalonii (<i>Pm</i>HMGR) catalyzes the oxidation of mevalonate and mevaldyl-CoA to form HMG-CoA using CoA-SH and two NAD+ cofactors. While the enzyme has been used extensively as a drug target in humans to treat hypercholesterolemia, its pathway has also been found to be critical for the survival of antibiotic resistant gram-positive bacteria. Structural studies using non-productive and slow-substrate binary complexes as well as biochemical studies using half and full reactions led to the proposal that the conversion of mevalonate to HMG-CoA occurs through the generation of two intermediates, mevaldehyde and mevaldyl-CoA (Shown in Fig 1.1). However, several intermediary changes along the <i>Pm</i>HMGR reaction pathway remain unclear. By gathering information about the enzyme’s intermediate states via structural studies, we could identify potential allosteric sites that further the reaction mechanism. Using this knowledge, we could design enzyme inhibitors that act as novel antibacterials. The application of time-resolved crystallographic methods would provide structural information about transitory states in the PmHMGR reaction mechanism. The <i>Pm</i>HMGR crystal has been shown to be suitable for time-resolved crystallographic measurements for the reaction steps resulting in mevaldyl-CoA formation. However, our structural investigations of the mevalonate, CoA and NAD+ complex that are expected to result in the formation of mevaldehyde (Fig 1.1) do not show any changes corresponding to a turnover in the crystal environment. <br></p><p><br></p><p>To investigate the factors limiting enzymatic activity in the crystal, we investigated the effects of pH and specific ions in the crystallization environment. Kinetic studies indicated a strong <i>Pm</i>HMHGR inhibition in the crystallization buffer that is dependent on the concentration of the crystallization precipitant ammonium sulfate. These studies also indicated an increase in enzyme turnover with increasing pH. Utilizing the ionic concentration and pH-dependent properties of the enzyme in the crystallization environment, we have developed a reaction triggering approach using pH changes for <i>Pm</i>HMGR crystals.<br></p><p><br></p><p>We have demonstrated our application of this ‘pH-jump’ method by observing changes in <i>Pm</i>HMGR crystals after reaction initiation. Changes in the density of mevalonate, CoA and NAD+have indicated mevaldehyde and mevaldyl-CoA formation. Additionally, the appearance of a unique NADH absorbance peak after the pH-change has also highlighted the initiation of the <i>Pm</i>HMGR reaction and the occurrence of a hydride transfer step. Our analysis of the movements using time-resolved structures post reaction-initiation have also highlighted structural changes and inter-domain contacts in the small and flap domain that would allow cofactor exchange and product release. The pH-jump method can hence be utilized as a novel approach for triggering the <i>Pm</i>HMGR reaction in crystals and further studying transitory states along its reaction pathway.<br></p>

Glutamate carboxypeptidase II (GCPII) is a zinc-dependent carboxypeptidase with high expression levels in prostate carcinoma. As the enzyme represents a validated target for cancer therapy and imaging, the development of new GCPII-specific ligands is still a focus of an active academic and industrial research. However, existing assays to screen inhibitor libraries and determine inhibitor efficacy are suboptimal at best. This thesis is aimed at the development of small internally quenched probes that could be used for continuous measurement of the GCPII enzymatic activity. These probes are derived from natural GCPII substrates and consist of a fluorophore/quencher pair connected by a GCPII-hydrolysable linker. I first characterized biophysical properties of the probes and then determined kinetic parameters of their hydrolysis by GCPII. The optimized activity assay was then used to determine inhibition constants of several GCPII-specific inhibitors. Finally, complexes between the inactive enzyme and several probes were co-crystallized and one of the complexes refined and analyzed. Our data show that the probes are involved in non-covalent interactions with the same amino acid residues of the enzyme's active site as natural substrates. The developed assay could be optimized for high-throughput...

Lincomycin is a naturally occurring member of a lincosamide group of antibiotics. The cluster of lincomycin biosynthetic gene was already decribed and the function of many of genes has been clarified. This work, "Characterization of N-demethyllincomycin-methyltransferase", is focused on the study of the final step of lincomycin biosynthetic pathway - the methylation of nitrogen atom from the pyrollo ring of the propylproline unit of the N-demethyllicomycin (NDL). The aim of this work was the characterization of the protein LmbJ, catalysing this final biosynthetic step. All the experiments were provided for the enzyme LmbJ with N-terminal histidine tag, which had been prepared by the heterologous expression in E.coli cells. The pH and temperature optimum was determined as well as the Michaelis constants for both substrates of the reaction - N-demethyllincomycin and S-adenosyl methionine (SAM - a methyl group donor). With the exception of the pH optimum, all specified parameters have markedly differed from the data published for the enzyme isolated from the natural source. Based on the comparison of electron microscopy, blue native gel electrophoresis and gel filtration results, the hypothetical model of the LmbJ quarternary structure was created. Majority of methyltranserases, so far described occure in...