A Thesis submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, for the Degree of Doctor of Philosophy. / A deficiency of the enzyme NADP dependent xylitol dehydrogenase (L-xylulose reductase) has previously been found to be the cause of chronic essential pentosuria. Essential pentosuria is a recessively inherited condition which is marked by the continual excretion of relatively large amounts of the enzymes substrate, L-xylulose. The major objective of the study described was to find a simple method for the identification of individuals who are heterozygous for the "pentosuria" and normal alleles. The pentosuria allele could then be used as a gene marker in linkage studies aimed at mapping the L-xylulose reductase locus. A L-xylulose reductase assay suitable for the identification of carriers of essential pentosuria was developed and tested on members of a South African Lebanese family in which the inheritance of pentosuria had previously been suggested to be dominant. It was found that family members could, on the basis of their L-xylulose reductase activities, be classified as either normal, heterozygous or homozygous for the pentosuria allele. Measurements of serum L-xylulose concentrations revealed that pentosuria is, contrary to the previous report, . recessively inherited in this family. A sample of the local Ashkenazi Jewish population was screened for pentosuria carriers. Six out of the 237 individuals screened were found (on the basis of their L-xylulose reductase activities and from the results of a loading test), to carry the pentosuria allele. The frequency of the pentosuria allele in this population was estimated from the apparent heterozygote frequency to be 0.0127. Linkage analyses were carried out on the families of the identified heterozygotes and on members of the Lebanese family mentioned above. No evidence of tight linkage was found between the pentosuria allele's locus and those coding for various red cell antigens, red cell enzymes and serum proteins. Kinetic, chromatographic and electrophoretic studies revealed that the red cells of normal individuals contain two distinct L-xylulose reductases, a minor and a major isozyme. Pentosurics lack the major isozyme but appear to have approximately normal amounts of the minor isozyme. The minor isozyme is e1ectrophoretica 1 1 y distinct from the major isozyme, has markedly higher Michael is constants for the substrates L-xylulose and xylitol and shows a lower pH optimum when catalysing the oxidation of xylitol. Electrophoresis also revealed that liver tissue contains two L-xylulose reductases which occur in similar proportions to those of red cells but which migrate at slightly different rates. / WHSLYP2016

Molecular drug design begins with the identication of a problem to solve. This work identies the growing resistance among human pathogens to current treatments. Once the problem is identied and understood, solutions must be proposed. This one is straight forward, we need new antimicrobial drugs. More specically, we need to identify novel targets to inhibit. A large portion of antibiotics focus on disruption of macromolecular production while only a few target metabolic systems. Finally, you need to propose solutions based on the information gathered. In order to avoid existing resistance, it is important to avoid the macromolecular route and focus on metabolic enzymes. Preferably, the pathway would have little overlap or similarity with pathways found in the treatment organism. With this in mind, the non-mevalonate (NMA) pathway poses as a very good target for drug design. Many pathogens have been found to be strictly dependent on this pathway while it is absent in humans. Additionally, fosmidomycin has already been shown to inhibit this pathway. Initially thought to just inhibit the 1-deoxy-D-xylulose 5- phosphate (DXP) reductoisomerase (DXR), it has been shown to inhibit several enzymes along the path to a lesser extent. Ideally, this could be repeated or improve upon for future drug design. With this in mind, the initial stages of the rst two enzymes of the NMA pathway were examined utilizing quantum mechanical/molecular mechanical (QM/MM) techniques. The rst enzyme was DXP synthase (DXS), which catalyzes a transketolase-like condensation of pyruvate and glyceraldehyde-3-phosphate to produce DXP. DXS and other transketolases are dependent on the thiamine diphosphate (TDP) cofactor, which must be deprotonated of the imidazolium C2 atom producing a highly reactive ylide. A tautomerization occurs prior to this deprotonation to prime the pyrimidinium ring N4 atom to perform the C2 abstraction. The question at hand was the identity of a general base to perform the N4 abstraction. The results favored a water-mediate mechanism with a higher than usual Ez of 22.7 kcal/mol. An observation pertaining the tautomerization pertained to the aromaticity of the pyrimidine ring. Upon further investigation, aromaticity was found to play a signicant role in the ΔE observed. Aromaticity might contribute 14.2 kcal/mol to the barrier height. This high energy would drive the reaction forward producing the ylide. Investigation of the DXR enzyme followed this work. Initially, the work was going to focus on the 2 mechanisms proposed for activity, alpha-ketol rearrangement and retroaldol/ aldol mechanism. Subsequent publications involving secondary kinetic isotope effects (KIEs) add to the pile of evidence supporting the retro-aldol/aldol mechanism. So the project was retooled to investigate the energetic dierences between two metal binding modes. The results of this work support a metal coordination across the C3-C4 bond, which eventually extends coordination to include the C2 oxygen. This conformation was help explain the tight binding eecting observation of the putative intermediates (transition states) and aldehyde intermediate. Additionally, as the C2-C3 mode consistently transfers a proton to the phosphate group of DXP or produces an elongated C-O bond, the C2-C3 mode would not be favorable. Further investigations of these enzymes (e.g. completing the step begin, continuing through the reaction) could provide further illumination into the mechanism of action and possibly reveal new avenues of drug design. Examining the enzymes downstream in the NMA pathway might provide details of interest. Of particular interest is the radical reaction proposed for HDR/IspH. The nal step of the pathway produces IDP and DMADP in a 4:1 proportion, which corresponds to the general system requirements for production of the long chain, branched isoprenoids. It would be interesting to compute the mechanism to see if energetics could provide further insights. Additionally, normal mode analysis coupled with vibrational subsystem analysis could identify allosteric sites for feedback sensitivity.

La voie non-mévalonate est fortement présente chez les plantes et les bactéries mais est absente chez les mammifères. C'est pourquoi inhiber la synthèse des isoprénoïdes et identifier un inhibiteur de cette voie enzymatique contribuera grandement à la recherche de nouveaux antibiotiques, antifongiques et herbicides. Les propriétés uniques de la 1-deoxy-D-xylulose 5-phosphate reductoisomérase (DXR), l'enzyme centrale de cette voie enzymatique, en font une cible très intéressante pour la synthèse de nouveaux composés. La Fosmidomycine agit comme un inhibiteur de la DXR et reste aujourd'hui, avec son homologue acétylé FR90098, la référence en termes d'inhibiteur même si de nombreux efforts ont été faits pour la synthèse d'analogues depuis plusieurs années comme expliqué dans le premier chapitre avec la mise en relation de la structure des composés et leur activité. L'analyse de la diffraction des rayons X de la DXR avec la Fosmidomycine où le substrat naturel montre que la fonction phosphonate ou phosphate interagit avec une poche polaire hautement spécifique dans le site actif de l'enzyme permettant peu de modifications. Par comparaison, la fonction acide hydroxamique qui chélate le cation de l'enzyme offre la possibilité de modifications par l'introduction d'autres fonctions complexantes. Dans ce contexte, de nombreuses modifications comme l'introduction de fonctions carbamoylphosphinate, amidoxime, N-hydroxyurée et dérivées d'uraciles comme unités complexantes ont été synthétisées pour trouver des nouvelles familles d'inhibiteurs de la DXR. Toutes ces fonctions possèdent des propriétés de chélation intéressantes. En effet, elles ont déjà conduit à de puissants inhibiteurs de différentes métalloenzymes. / The non-mevalonate pathway is highly present in higher plants, protozoa and bacteria but as no equivalent in mammals. That is why shut down isoprenoid biosynthesis and identify a non-mevalonate pathway inhibitor would greatly contribute to the search for safer antibiotics, antimalarials and for our concern herbicides. The unique properties of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), the central enzyme of this pathway, make it a remarkable and attractive target for drug design. Fosmidomycin acts as an inhibitor of DXR and still remains, along with its N-acetyl homologue FR90098, one of the most potent inhibitor ever known even if extensive work on the development of Fosmidomycin analogue derivatives have been developed since the last decade as demonstrated in the first chapter with the development of a structure activity relationship of all the potential inhibitors of this enzyme already reported in the literature. The X-ray diffraction analysis of the co-crystals of DXR and Fosmidomycin or substrate shows that the phosphonic/phosphate group interacts with a highly specific polar pocket in the enzyme site, allowing only few structural modifications. By contrast, the cation chelating subunit represented by the hydroxamic acid function offers fine tuning possibilities for the complexation abilities as well as potential secondary interactions with the NADPH cofactor or directly with the enzyme. In this context, several modifications such as the introduction of carbamoylphosphinate, amidoxime, N-hydroxyurea and uracil complexing subunits have been made in order to find new families of DXR inhibitors. All of these functions show promising chelation capabilities as they already led to potent inhibitors of different metalloenzymes.

Ce travail pluridisciplinaire de thèse présente un éclairage sur le mécanisme de méthylation enzymatique de triterpénoïdes de la famille du hopane. Au cours de ce travail, nous nous sommes intéressés à déterminer la stéréochimie de la réaction enzymatique de méthylation en C-2 du cycle A du hopane. Cette stéréochimie a été étudiée par vérification de la rétention ou non du deutérium provenant du (5R)- et du (5S)-(5-2H1)-1-désoxy-Dxylulose, incubés dans des cellules bactériennes produisant des triterpénoïdes méthylés en C-2. Après avoir effectué la synthèse des deux isotopomères (R) et (S) marqués audeutérium en C-5 du DX, nous les avons incorporés dans les triterpénoïdes méthylés en C-2 chez Bradyrhizobium japonicum. Nous avons pu montrer par analyse en RMN 13C de ces triterpénoïdes méthylés isolés que la méthylation en C-2 n’est pas totalement stéréosélective, mais majoritairement une substitution du proton pro (R) en C-2 par le groupement méthyle a eu lieu. / This multidisciplinary thesis presents an insight into the mechanism of some enzymatic methylation of triterpenoids of the hopane family. In this work, we are interested in determining the stereochemistry of the enzymatic methylation reaction on C-2 of the hopane ring. This stereochemistry was studied by checking the retention or the non retention of the deuterium atom provided from (5R)- and (5S)-(5-2H1)-1-deoxy-D-xylulose incubated in bacterial cells producing triterpenoids methylated on C-2. After achieving the synthesis of the two isotopomers (R) and (S) deuterium-labeled on C-5 of the 1-deoxy-Dxylulose, we proceed to incorporate these two isotopomers of the DX into the methylated triterpenoids on C-2 isolated from Bradyrhizobium japonicum. 13C NMR analysis of these methylated triterpenoids showed that methylation on C-2 is not completely stereoselective, but mostly the pro (R) proton on C-2 is substituted by the methyl group.

No description available.

La synthèse enzymatique de terpénoides chez les mammifères provient de la voie mevalonique. Récemment une voie différente a été découverte et s'est révélée être prépondérante pour de nombreux organismes comme les plantes et bactéries. L'identification d'un inhibiteur de cette cascade enzymatique permettrait le développement d'une nouvelle famille d'herbicide. Les caractéristiques de la 1-déoxy-D-xylulose 5-phosphate réductoisomérase (DXR) font de cette enzyme très spécifique une cible pour la synthèse de nouveaux composés. La Fosmidomycine ainsi que son analogue acétylé le plus proche, FR-900098 restent des références pour l'inhibition de la DXR. Dans ce contexte, l'ensemble des molécules décrites dans la littérature en tant qu'inhibiteurs a été classé en fonction des modifications apportées sur le substrat naturel ou la Fosmidomycine. A partir de l'ensemble de ces informations, cinq familles ont été synthétisées pour trouver un nouveau motif complexant. Pour deux d'entre elles, le squelette de base contient un acide phosphonique et un acide phosphinique sur lequel a été introduit la diversité moléculaire grâce aux réactions de Pudovik et de couplage pallado-catalysé. Les autres motifs complexant originaux sont constitués d'une fonction carbonyle et d'un hétérocycle en α ou β. Après optimisation de la synthèse des précurseurs, la diversité a été introduite à l'aide, par exemple, d'une réaction de trois composantes permettant la préparation d'hétérocycle. Enfin, deux modifications ont été faites sur le bras espaceur : l'introduction d'atomes de fluor pour modifier les propriétés physicochimiques ou d'un atome d'azote, point d'attache de nouveaux groupements. / The non-mevalonate pathway is widely found in higher plants and in many eubacteria, including pathogenic ones, but not in mammals. Identifying a non-mevalonate pathway inhibitor would greatly contribute to the search for new herbicides. The unique properties of 1-Deoxy-D-xylulose 5-phosphate reductoisomerase make it remarkable and rational target for drug design. The phosphonohydroxamic acid Fosmidomycin, which acts through inhibition of DXR, is a natural compound produced in the fermentation of Streptomyces and still remains, with its N-acetyl homologue FR900098, one of the most active compounds. First of all, the enzyme and all the potential inhibitors tested in literature were classified in order to understand the global quest for therapeutically useful compounds. In this context, we designed and synthesized five different families of Fosmidomycin analogues containing a new chelating unit. Two targets molecules families bearing a phosphinophonic acid as common core were imagined. Divergent approach allowed the introduction of the chemical diversity thank to powerful pallado-catalyzed coupling reaction. The other families containing carbonyl group and heterocycle in α‐ and β‐position were regarded as highly potent complexing units. Chemical diversity was introduced mainly at the end of the synthesis. For one of them convergent ring formation using three-components reaction was developed. Finally two modifications of the Fosmidomycin linker were performed by the introduction of fluorine atoms on the parent structure as well as the replacement of a carbon by a nitrogen atom in order to create a new point of modifications.

Isoprenoids are the largest family of biologically active compounds, synthesized by five carbon subunits namely isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). For long time the mevalonate-dependent (MVA) pathway has been considered as the sole source of IPP and DMAPP, until recently a new non-mevalonte dependent (NMVA) pathway was discovered. This new pathway utilizes entirely different set of enzymes for isoprenoids synthesis and don't have any homologues in humans. NMVA pathway is the only source of isoprenoids for certain eubacteria, parasite and plants. Absence of the NMVA pathway in higher organisms has opened a new platform for the development of novel antibiotics and antimalarials. 1-deoxy-D-xylulose-5-phosphate synthase (DXS), the first enzyme in NMVA pathway has been reported as the rate limiting enzyme in the synthesis of IPP and DMAPP and has been the center of interest for inhibitor development. Reaction mechanism of thiamine pyrophosphate (TPP) and Mg2+ dependent DXS enzyme has been studied in this report. Using steady state kinetics analysis, product inhibition and dead end inhibitor, the mechanism of substrate (pyruvate and D-glyceraldehyde-3-phosphate) addition was studied. Due to different domain organization in DXS as compared to theother TPP dependent enzyme, the mechanism of addition was found to be random sequential rather than ping-pong mechanism. Based on bioinformatics tool and in vitro studies it has been established that NMVA exists in all the plasmodium species, thus making the enzymes involved in NMVA as an alluring target for new antimalarial drugs. All the plasmodium species and other member of the phylum apicomplexa harbor apicoplast an organelle which is homologous to the chloroplast of plants and algae. All the enzymes from NMVA pathway translocate to apicoplast from nucleus through a secretory pathway using signaling and transit peptide. In this study DXS from P. vivax has been cloned and expressed in E. coli using genomic DNA and codon optimized synthetic DNA as a source. Expression of full length DXS with signal and transit peptide as well as mature protein without these peptide using serial deletion has been studied. Kinetic parameters of P.vivax DXS have been calculated and found to be comparable to the DXS from other species.

No description available.

Mycobacterium tuberculosis, the causative pathogen of tuberculosis, currently infects one-third of the world’s population, resulting in two million deaths annually. This clearly shows that tuberculosis is one of the most serious diseases of our times. The often unpleasant side effects from the current drugs, combined with the difficulty of ensuring patient compliance, and the emergence of drug-resistant and multidrug-resistant strains, makes the need for new and better drugs urgent. In this thesis, all the steps, from cloning, purification, crystallization, to activity determination, and structure determination are presented for two different M. tuberculosis enzymes. The structures, which were modeled from X-ray crystallographic data, provide the framework for structure-based drug design. Here, new potential inhibitors can be tailor-made based on the specific interactions in the enzyme’s active site. The bacteria have two different peptidyl-prolyl cis-trans isomerases that catalyze the isomerization of peptide bonds preceding proline residues, a process of high importance for correct folding. Here we present the structure of peptidyl-prolyl cis-trans isomerase A, an enzyme present inside the bacteria, and distinguish it from the B form of the enzyme, which is membrane bound, placing its active site outside the bacteria. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step within the non-mevalonate pathway, which leads to the production of isopentenyl diphosphate. This compound is the precursor of various isoprenoids, vital to all living organisms. In humans, isopentenyl diphosphate is produced via a different pathway, indicating that all the enzymes within the non-mevalonate pathway may be suitable drug targets in M. tuberculosis. Several structures of both wild type and mutant 1-deoxy-D-xylulose-5-phosphate reductoisomerase in complex with different substrates, and also with the known inhibitor fosmidomycin, provide valuable information not only to the field of drug design, but also, in this case, into the catalysis.

Made available in DSpace on 2016-06-02T19:56:52Z (GMT). No. of bitstreams: 1 5322.pdf: 3668179 bytes, checksum: e3612c159bebd3f0a5be1640868316ae (MD5) Previous issue date: 2013-03-21 / Financiadora de Estudos e Projetos / The conversion of the hemicellulosic fraction in ethanol is a factor that impacts on the economic viability of the second generation ethanol production process from sugar cane bagasse. Hemicellulose from bagasse is a heteropolymer constituted by pentoses and glucose, being xylose the predominant sugar (~ 21 %). Among the available technological alternatives for ethanol production from xylose, SIF process (Simultaneous Isomerization and Fermentation), consisting of xylose conversion to xylulose by glucose isomerase (GI) enzyme and xylulose fermentation by the yeast S. cerevisiae, is considered a promising alternative. The main objectives of the present work were: i) evaluate the performance of different S. cerevisiae strains towards xylulose intake and ethanol productivity; ii) assess the influence of cultivation conditions (temperature, oxygen availability and initial xylose concentration) upon ethanol and xylitol production by the selected strains; iii) define the operation conditions for the continuous SIF process, using a system of fixed bed reactors associated in series. Preliminary experiments were conducted in 50 mL flasks, containing 4 g of pellets with a load of 20 % of immobilized GI, co-immobilized with yeast (load of 10 %) in alginate gel. For the screening of yeasts showing better performance on ethanol production from xylose, two commercial baker´s yeast strains (Itaiquara® e Fleischmann®), three industrial strains (BG-1, CAT-1 e PE-2) and one lab strain (CEN.PK113-7D) were evaluated. These experiments were performed at 35 oC, using a medium composed by xylose (60 g/L), urea (5 g/L), CaCl2 (1.9g/L) and several salts, at initial pH of 5.6. Additional SIF studies were carried out with the selected yeasts Itaiquara®, BG-1 or CEN.PK113-7D under different temperature conditions (40 oC), aeration (15 mL flasks) and initial xylose concentration (130 g/L) for comparison with the results obtained at the standard conditions. For SIF cultures, samples were withdrawal and the concentrations of reducing sugars were determined by DNS method while xylose, xylulose, ethanol and by-products (xylitol, glycerol etc) concentrations were assessed by liquid chromatography. Cell viability was also measured at the beginning and end of the experiment. When comparing the different yeasts, Itaiquara® strain presented the best performance, reaching ethanol concentrations of 22.4 g/L, with a productivity of 2.1 g/Lh. The conversion of xylose was similar for all studied industrial strains as well as among the baker s yeast and lab strains. Concerning the group of additional experiments, at 40 oC, a decrease of viability and ethanol selectivity was observed for Itaiquara®, whereas productivity and selectivity for CEN.PK113-7D. was improved. For the studies conducted under semianaerobic conditions, the yeast BG-1 showed an increase in selectivity and yield. However, the reaction time increased to app. 45 hours. On the other hand, the performance of strain Itaiquara® was not altered by the lower level of oxygen tested. In the experiment with 120 g/L of xylose, more than 40 g/L of ethanol was obtained in 24 hours of cultivation. Thus, we conclude that the SIF process proposed in the present work is a viable alternative for the production of ethanol from xylose or lignocellulosic residues. For the operation of the continuous system composed by fixed bed reactors associated in series, the recommended conditions include the Itaiquara® yeast with a temperature no higher than 35 oC, keeping the total residence time around 10 hours for a feeding supply containing 60 g/L of xylose. / A conversão da fração hemicelulósica da biomassa em etanol é um dos fatores que impactam a viabilidade econômica do processo de produção de etanol de segunda geração a partir do bagaço de cana-de-açúcar. A hemicelulose do bagaço é um heteropolímero constituído por pentoses e glicose, sendo a xilose o açúcar predominante (~ 21 %). Dentre as diversas alternativas tecnológicas para a produção de etanol a partir de xilose, o processo SIF (Simultânea Isomerização e Fermentação), consistindo na isomerização da xilose em xilulose pela enzima glicose-isomerase (GI) e na fermentação da xilulose pela levedura S. cerevisiae, é considerado uma alternativa promissora. Os principais objetivos do presente trabalho foram: i) avaliar o desempenho de diferentes cepas de S. cerevisiae em termos de assimilação de xilulose e produtividade em etanol; ii) estudar a influência das condições de cultivo (disponibilidade de oxigênio, temperatura e da concentração inicial de xilose) na produção de etanol e xilitol pelas cepas selecionadas; iii) definir as condições de operação para um processo SIF contínuo em sistema de reatores de leito fixo associados em série. Os experimentos preliminares foram conduzidos em frascos de 50 mL contendo 4 g de pelletes com carga de 20 % de glicose isomerase imobilizada, coimobilizada com levedura (carga de 10%) em gel de alginato. Para a seleção da levedura com melhor desempenho na produção de etanol a partir de xilose, foram avaliadas duas linhagens de levedura de panificação comercial (Itaiquara® e Fleischmann®), três cepas industriais (BG-1, CAT-1 e PE-2) e uma utilizada em laboratório (CEN.PK113-7D). Esses experimentos SIF foram conduzidos a 35ºC utilizando meio composto por xilose (60 g/L), ureia (5 g/L), CaCl2 (1,9 g/L) e sais diversos, em pH inicial 5,6. Experimentos SIF complementares foram realizados com as leveduras selecionadas Itaiquara®, BG-1 ou CEN.PK113-7D em diferentes condições de temperatura (40oC), aeração (frascos de 15 mL) e concentração inicial de xilose (130 g/L) para comparação com os resultados obtidos nas condições padrão. Em todos os experimentos SIF, amostras foram retiradas para determinação da concentração de açúcares redutores (método DNS) e de xilose, xilulose, etanol e subprodutos (xilitol, glicerol etc.) por cromatografia em fase líquida. Foi também acompanhada a viabilidade celular ao longo do cultivo. Na comparação entre as diferentes leveduras, destacou-se especialmente a levedura Itaiquara®, alcançando concentrações de etanol de 22,4 g/L, com produtividade em etanol de 2,1 g/Lh. A conversão de xilose foi semelhante entre as leveduras industriais e entre as leveduras de panificação e a de laboratório. Quanto ao conjunto de experimentos complementares, na temperatura de 40ºC houve diminuição de viabilidade e seletividade em etanol para a Itaiquara® e melhora na produtividade e seletividade para a CEN.PK113-7D. Nos experimentos realizados em condições semianaeróbias, a levedura BG-1 apresentou aumento de seletividade e rendimento em etanol, porém para um tempo de reação de 45 horas, aproximadamente. Já a levedura Itaiquara® não teve seu desempenho influenciado pela menor disponibilidade de oxigênio. No experimento realizado com 130 g/L de xilose, alcançou-se mais de 40 g/L de etanol em 24 horas de cultivo. Conclui-se, assim, que o processo SIF de xilose, proposto no presente trabalho, é uma alternativa viável para a produção de etanol a partir de xilose ou de resíduos lignocelulósicos. Para a operação em sistema contínuo composto por reatores de leito fixo associados em série recomenda-se a utilização de levedura Itaiquara® e de temperatura de, no máximo, 35ºC, mantendo-se o tempo de residência total em torno de 10 horas para uma alimentação contendo 60 g/L de xilose.