Pantothenate (vitamin B5), which is the invariable metabolic precursor to coenzyme A, is synthesized from L-aspartate and alpha-ketoisovalerate in a converging four-step process in bacteria. Here, structural studies of two enzymes of pantothenate biosynthesis in Escherichia coli, L-aspartate-alpha-decarboxylase and ketopantoate hydroxymethyltransferase, are described. Ketopantoate hydroxymethyltransferase catalyzes the transfer of a hydroxymethyl group on to alpha-ketoisovalerate, assisted by the cofactor 5,10-methylene-5,6,7,8-tetrahydrofolate. In order to determine the mode of cofactor binding to the protein, ketopantoate hydroxymethyltransferase was crystallized in the presence of two 5,10-methylene-5,6,7,8-tetrahydrofolate analogues and alpha-ketoisovalerate. X-ray diffraction patterns, collected on the in-house X-ray diffraction data collection facility, extended to 4.0 Angstroem. Unit cell dimensions derived from these diffraction patterns indicate an asymmetric unit with one decameric enzyme. A detailed comparative structural analysis of the fold of ketopantoate hydroxymethyltransferase was carried out. Based on this investigation it was possible to assign the enzyme to the phosphoenolpyruvate/pyruvate enzyme superfamily. Furthermore, similarities in the mode of ligand binding to the catalytic magnesium, as well as differences in the mechanisms between the enzymes within this superfamily could be delineated. In common with a small, but widely distributed, group of mechanistically-related enzymes, L-aspartate-alpha-decarboxylase is translated as an inactive pro-enzyme, which self-processes at a specific site. In this process of intra-molecular protein maturation a covalently bound pyruvoyl cofactor is formed. A fast purification system for eight L-aspartate-alpha-decarboxylase mutants was established that allows production of large amounts of enzyme. In order to gain insights into the molecular mechanism of self-processing, crystallographic studies were carried out. Several of the purified mutants have been crystallized. X-ray diffraction data from glycine 24 to serine and serine 25 to threonine mutants were collected, to a maximum resolution of 1.26 Angstroem. The respective crystal structures were solved by molecular replacement. Along with the structures of an unprocessed, native precursor form of L-aspartate-alpha-decarboxylase and a serine 25 to alanine mutation, the structure models were refined and evaluated, and the models deposited in the Protein Data Bank. Analysis of these four structures together with four other L-aspartate-alpha-decarboxylase mutant structures revealed specific conformational constraints on the self-processing mechanism. Threonine 57 and a water molecule could be identified as catalytic elements, most likely essential for acid-base catalysis, and stabilization of the oxyoxazolidine intermediate in the self-processing reaction. A molecular mechanism for self-processing in L-aspartate-alpha-decarboxylase, largely based on the threonine 57 and a water molecule, is proposed. The differences in the structures of the cleavage site of the serine 25 to alanine and serine 25 to threonine mutants, relative to the structure of the unprocessed native precursor, suggest that molecular models of the cleavage site and mechanisms, based solely on serine to alanine and serine to threonine mutants, may lead to erroneous interpretations of the mechanism. On comparison with other self-processing systems, particularly, glycosylasparaginase, remarkable parallels in the structural features of the environment of the cleavage site were identified.

Malaria is prevalent in over 100 countries which is populated by half of the world’s population and culminates in approximately one million deaths per annum, 85% of which occurs in sub-Saharan Africa. The combined resistance of the mosquitoes and parasites to the currently available pesticides and antimalarial chemotherapeutic agents requires the concerted effort of scientists in the malaria field to identify and develop novel mechanisms to curb this deadly disease. In this study, a thorough understanding of the role players in the polyamine pathway of the parasite was obtained, which could aid future studies in the development of novel inhibitory compounds against these validated drug targets. The uniquely bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) of Plasmodium falciparum forms an important controlling node between the polyamine and methionine metabolic pathways. It has been speculated that the unique bifunctional association of the rate-limiting enzymes allows for the concerted regulation of the respective enzyme activities resulting in polyamine synthesis as per requirement for the rapidly proliferating parasite while the methionine levels are strictly controlled for their role in the methylation status. The results of this study showed that the enzyme activities of the bifunctional complex are indeed coordinated and subtle conformational changes induced by complex formation is suggested to result in these altered kinetics of the individual AdoMetDC and ODC domains. Studies also showed that the identification of the interaction sites between the domains, which allows for communication across the complex, may be targeted for specific interference with the enzyme activities. Furthermore, these studies showed that the current knowledge on the different subclasses of the AdoMetDC family should be re-evaluated since P. falciparum AdoMetDC shows diverse properties from orthologues and therefore points towards a novel grouping of the plasmodial protein. The extensive biochemical and biophysical studies on AdoMetDC has also provided important avenues for the crystallisation and solving of this protein’s 3D structure for subsequent structure-based identification of drug-like lead compounds against AdoMetDC activity. The application of structure-based drug design on malarial proteins was additionally investigated and consequently proved that the rational design of lead inhibitory compounds can provide important scaffold structures for the identification of the key aspects that are required for the successful inhibition of a specific drug target. Spermidine synthase, with its intricate catalytic mechanism involving two substrate binding sites for the products of the reactions catalysed by AdoMetDC/ODC, was used to computationally identify compounds that could bind within its active site. Subsequent testing of the compounds identified with a dynamic receptor-based pharmacophore model showed promising inhibitory results on both recombinant protein and in vitro parasite levels. The confirmation of the predicted interaction sites and identification of aspects to improve inhibitor interaction was subsequently investigated at atomic resolution with X-ray protein crystallography. The outcome of this doctoral study shows the benefit in applying a multidisciplinary and multinational approach for studying drug targets within the malaria parasite, which has led to a thorough understanding of the targets on both biochemical and structural levels for future drug design studies. / Thesis (PhD)--University of Pretoria, 2011. / Biochemistry / unrestricted

Porphyria is a group of inherited metabolic disorders due to enzymatic defect of the heme biosynthesis resulting in the overproduction of the heme precursors' porphyrins in different body organs. The enzymes of the heme biosynthesis are encoded by corresponding genes in which any defect in any of these genes lead to a specific type of porphyria. Numerous mutations were detected in these genes leading to impairment in the enzyme function and therefore developing of the clinical manifestations of porphyria. The aim of the present work was to investigate the UROD gene in patients with porphyria cutanea tarda (PCT) and hepatoerythropoietic protoporphyria (HEP) as well as the FECH gene in patients with erythropoietic protoporphyria (EPP) on a molecular level. We identified numerous mutations in the FECH and the UROD genes in three different populations, Czech, Slovak, and Egyptian. We described the novel mutations in the UROD gene in HEP Arabic patients from Egypt as well in the FECH gene in patients with EPP of Czech and Slovak origin. We expressed mutatted UROD protein in prokaryotic system and found 19 % of the wild-type enzymatic activity. Moreover, the current study presents for the first time the frequency of the low expression allele IVS3-48c in the FECH gene in healthy controls from the Czech...

Growing concerns of the negative effects on the environment and dependency of fossil fuelsare major driving forces for finding novel sustainable production pathways for plastic.Metabolic engineering has emerged as a powerful tool to enable microorganisms to producenon-native metabolites. The aim of this project was recombinant production of 4-hydroxybutyrate (4-HB) by expressing two enzymes in the model organism Escherichia coli.α-ketoglutarate decarboxylase (SucA) from Mycobacterium smegmatis followed by 4-hydroxybutyrate dehydrogenase (4-HBd) from Clostridium kluyveri was expressed inEscherichia coli. Results showed that the genes were successfully transformed and expressedin E. coli and after protein purification a concentration of 0.9 g/L SucA and 9.8 g/L 4-HBdwas achieved. Furthermore, some protein activity was detected by a coupled reaction withSucA and 4-HBd. When the enzymes got coupled together a change in NADH concentrationcould be detected spectrophotometrically. The enzymes were also tested for substratespecificity by using substrates with various carbon chain lengths and a decrease in NADHconcentration was seen. However, a decrease in the negative control for the experiments wasalso seen indicating a breakdown of NADH over time rather than consumption. Therefore, noconclusion could be drawn about the promiscuity of the enzymes. Lastly a single plasmidssystem was tested where both the genes were ligated on the same plasmid (pCDF duet) andexpressed successfully in E. coli Bl21DE3. / Ökad oro för miljön samt behovet av fossila resurser för produktion av plaster har gjort detnödvändigt att skapa nya och mer hållbara produktions vägar. Genetisk modifikation av olikaorganismer har utvecklats som ett starkt redskap för att få mikroorganismer att framställametaboliter som de normalt inte producerar. Målet med detta projekt var rekombinantproduktion av gamma hydroxibutansyra (4-HB) genom att uttrycka två enzym i modellorganismen Escherichia coli. Dessa enzym bestod av α-ketoglutarat dekarboxylas (SucA) frånMycobacterium smegmatis samt 4-hydroxybutyrate dehydrogenas (4-HBd) från Clostridiumkluyveri. Resultaten visade att proteinerna lyckades utryckas i E. coli med en koncentration av0,9 g/L SucA och 9,8 g/L 4-HBd som uppnåddes efter rening. Utöver detta detekterades ävenviss enzymaktivitet genom att kopplad enzymreaktion mellan 4-HBd och SucA och mätakonsumtionen av NADH spektrofotometriskt över tid. Enzymen testades även försubstratspecificitet genom att köra reaktionen med substrat med olika längd på kolkedjan. Dåkunde en minskning i NADH koncentrationen ses men det gjordes det även för de negativakontrollerna vilket indikerar nedbrytning av NADH och inte konsumtion av NADH. Ingaslutsatser angående enzymens substratspecificitet kunde därför dras. Det sista som gjordes varatt sätta in båda generna i ett en plasmidsystem där båda generna sattes in på samma plasmid(pCDF duet) och uttrycktes framgångsrikt i E. coli Bl21DE3.

Background: Type 1 Diabetes mellitus (TIDM) is a chronic disorder in which the insulin producing beta cells are selectively self-destroyed. Although the etiology of the disease has not been determined, genetic dispositions such as SLC11A1 polymorphism in suffering patients have been reported. The role of pathogenic microorganisms such as Mycobacterium avium subspecies paratuberculosis (MAP) in TIDM has also been recently debated. MAP is already known to cause paratuberculosis in cattle and now it is a strong suspect of causing autoimmune diseases in humans such as Crohn’s disease, multiple sclerosis, autoimmune Thyroiditis, rheumatoid arthritis and autoimmune diabetes. We hypothesis that molecular mimicry between MAP Heat shock protein 65K (Hsp65) and human Glutamic Acid Decarboxylase 65K (GAD65) can be the trigger which leads to the autoimmune destruction of beta cell in patients exposed to MAP . Method: To test the hypothesis, peptide sequences of MAP Hsp65 and human GAD65 were investigated using BLAST and PyMOL bioinformatics tools. Moreover, 18 blood samples from humans with TIDM and controls, and 100 sera samples from cattle with paratuberculosis and controls were evaluated for the presence of MAP, MAP DNA and its antibodies. Glucose, insulin and GAD65 antibodies were also determined in some of the clinical samples. Results: Peptide BLAST analysis revealed 44% identity between the two proteins with 75% positive identities in a 16 amino acid region. PyMOL structural analysis identified possible shared epitope regions of the proteins in its 3D conformation. Immunoblot analysis revealed a strong cross reactivity between lysate of E.coli recombinant of MAP Hsp65 and plasma from human subject with TIDM. A weak cross reactivity was also observed between healthy rat pancreatic homogenate and rabbit anti MAP IgG. Nested PCR using IS900-specific iv oligonucleotide primers did not detect MAP DNA in peripheral blood from 18 subjects with Type I Diabetes, Type II Diabetes and non-diabetic controls. Long term culture of leukocytes from blood samples from same subjects resulted in the presence of MAP in 3/10 (30%) TIDM and 4/8 (50%) control subjects. However anti MAP IgG were detected in 5/10 (50 %) TIDM samples compared to 3/8 (37.5 %) controls. Insulin level was measured in sera from paratuberculosis cattle and controls. In MAP infected cattle, insulin level ranged from below 0.1ng/ml to 2.456 ng/ml with an average of 0.36 +/- 0.57ng/ml compared to below 0.1ng/ml to 13.47ng/ml with an average of 2.86 +/- 3.00ng/ml in healthy cattle. Conclusion: Bioinformatics analysis between MAP Hsp65 and human GAD65 through BLAST and PyMOL analysis revealed a homology of 16 amino acid motif and possible shared epitope regions; immunohistochemistry analysis revealed a cross reactivity between rabbit antiMAP IgG and pancreatic cell homogenate from a healthy rat. Moreover, plasma from patient with TIDM (TD8), who was confirmed to be positive for MAP DNA and MAP IgG, reacted strongly with MAP Hsp65 in MAP protein lysate and MAP Hsp65 recombinant clone pmptb20. Culture of MAP from human leukocytes is significant despite the lack of correlation between MAP in samples from TIDM and controls. It is worth noting that some of the control subjects have not been evaluated for other autoimmune diseases possible MAP role. Additionally, antiMAP IgG levels in TIDM subjects compared to controls have raised a suspicion on the involvement of MAP in TIDM. The poor correlation of MAP in blood versus either the antiMAP IgG or the insulin level may be related with the fastidious nature of MAP and in vitro cultivation. Since MAP is the sole causative agent of Johne’s disease, it is significant that the insulin level is 8 folds less in MAP infected cattle compared to MAP free cattle. Overall, the data v is mixed and suggest that further study is needed to investigate the intriguing question to whether MAP is involved in TIDM or not.

No description available.

A parede celular vegetal, estrutura essencial para as plantas, é extremamente importante para a economia humana, já que apresenta diversas utilidades, como por exemplo, fabricação de papel, fibras de vestuário, construção civil, entre outras. A maior parte da parede celular vegetal primária (aproximadamente 90%), é formada por polissacarídeos como celulose, hemiceluloses e pectinas. Os monossacarídeos, unidades formadoras dos polissacarídeos, são sintetizados, nas plantas, a partir de diferentes açúcares nucleotídeos, sendo que, o suprimento desses, pode afetar a biossíntese dos polissacarídeos da parede celular. Visando analisar o impacto da alteração do fluxo metabólico do carbono na composição da parede celular, o presente projeto de pesquisa teve como objetivo alterar a composição dos polissacarídeos da parede celular de Nicotiana tabcum, através da modulação da expressão do gene uxs, responsável pela codificação da enzima UDP-D-glucuronato descarboxilase (UDPGlcADC, EC 4.1.1.35) que converte UDP-D-glucuronato em UDP-D-xilose, importante açúcar nucleotídeo, precursor do monossacarídeo xilose. Para isso, após a clonagem do gene uxs de ervilha, foram obtidas plantas transgênicas de tabaco superexpressando esse gene. Diversas análises foram realizadas para determinação da composição química da parede celular primária e secundária dessas plantas. Pela análise de FTIR da parede celular primária, verificou-se que três linhagens transgênicas apresentaram espectrotipos consistentes, indicando uma redução na quantidade de pectinas e ligações ésteres carboxílica nessas linhagens transgênicas. Apesar de não terem sido detectadas alterações na proporção dos monossacarídeos ramnose, xilose, arabinose, manose e galactose, e na quantidade de celulose, na parede celular primária das plantas transgênicas, foram observadas diferenças na proporção de galactose não esterificada, nas linhagens que apresentaram espectrotipo. Com relação à parede celular secundária, observou-se que algumas linhagens transgênicas apresentaram maior concentração de lignina solúvel relacionada a uma redução no conteúdo de lignina insolúvel. / The plant cell wall is not only an essential structure for plants, but also an extremely important raw material in human economy. The plant cell wall has diverse utilities, for example, papermaking, textile fiber, civil construction. Polysaccharides, such as cellulose, hemicelluloses and pectins, are the major components of the primary plant cell wall (approximately 90%). These polysaccharides are formed by monosaccharides, which are synthesized in the plant from different nucleotide sugars. The suppliment of the nucleotide sugars can affect plant cell wall polysaccharides biosynthesis. Aiming at analyzing the impact of the alteration in the metabolic carbon flux on cell wall composition, the objective of this research project was to alterate the plant cell wall polysaccharides composition by the modulation of the uxs gene. This gene encodes the UDP-D-glucuronic acid decarboxylase enzyme (UDPGlcADC, EC 4.1.1.35) that promotes the conversion of UDP-D-glucuronic acid to UDP-D-xylose, an important sugar nucleotide precursor of xylose monosaccharide. To achieve this goal, the pea uxs gene was cloned and transgenic tobacco plants overexpressing this gene were obtained. Several analyses were performed to determinate the primary and secondary cell wall composition of those transgenic plants. The primary cell wall analysis by FTIR identified three transgenic lines that show different spectrotypes compared to wild type and those transgenic spectrotypes had the same features. The results indicate a reduction of pectin and ester carbonyl binding in the transgenic plants. No alterations were detected in the monosaccharide (rhamnose, xylose, arabinose, manose and galactose) proportions and the amount of cellulose in the primary cell wall of the transgenic plants. Nevertheless, differences in the proportion of unesterified galactose were observed in the same transgenic lines that showed spectrotypes. With regard to secondary cell wall, some transgenic lines showed an increase in soluble lignin which is related to a reduction in insoluble lignin.

<p>Organ-specific autoimmunity constitutes a large health problem, where both the clinical management and our understanding of the pathogenetic mechanisms need to improve. Women with Addison's disease have abnormally low levels of dehydroepiandrosterone (DHEA), its sulphate ester (DHEA-S) and androgens relative to age, and many patients complain of physical and mental fatigue and low stress tolerance. To define a suitable dose, the effect of oral DHEA replacement was evaluated in women with Addison's disease. </p><p> DHEA was administered for three months to nine women with Addison's disease in either of two doses, 50 mg (n=5) or 200 mg (n=4). A dose of 50 mg restored the DHEA(S) and androgen levels to normal without altering the insulin sensitivity, body composition or serum lipid profile.</p><p> Autoimmune polyendocrine syndrome type I (APS I) is a rare but useful model disorder of autoimmunity, characterised by multiple organ-specific autoimmune manifestations and high-titre autoantibodies and with adrenocortical insufficiency, Addison's disease, as one of its cardinal manifestations. Approximately 10-20% of APS I patients suffer from autoimmune hepatitis, which carries a high mortality, if untreated. The presence of putative antigenic targets in the liver was investigated.</p><p> Cytochrome P4501A2 (CYP1A2) and aromatic L-amino acid decarboxylase (AADC) were identified as hepatic autoantigens with the use of APS I sera for immunofluorescent staining of normal human liver, Western blot of microsomal and cytosol fractions of human liver homogenate, and immunoprecipitation of <i>in vitro</i> transcribed and translated radioactively labelled proteins. The presence of CYP1A2- and AADC-antibodies was significantly correlated to AIH, and CYP1A2 antibodies inhibited enzyme activity <i>in vitro</i>.</p><p><i> In conclusion</i>, a daily replacement dose of 50 mg of DHEA sufficiently restores levels of DHEA, DHEA(S) and androgens in women with Addison's disease, without severe side-effects. We have further identified CYP1A2 and AADC as hepatic autoantigens associated with autoimmune hepatitis in APS I.</p>

Organ-specific autoimmunity constitutes a large health problem, where both the clinical management and our understanding of the pathogenetic mechanisms need to improve. Women with Addison's disease have abnormally low levels of dehydroepiandrosterone (DHEA), its sulphate ester (DHEA-S) and androgens relative to age, and many patients complain of physical and mental fatigue and low stress tolerance. To define a suitable dose, the effect of oral DHEA replacement was evaluated in women with Addison's disease. DHEA was administered for three months to nine women with Addison's disease in either of two doses, 50 mg (n=5) or 200 mg (n=4). A dose of 50 mg restored the DHEA(S) and androgen levels to normal without altering the insulin sensitivity, body composition or serum lipid profile. Autoimmune polyendocrine syndrome type I (APS I) is a rare but useful model disorder of autoimmunity, characterised by multiple organ-specific autoimmune manifestations and high-titre autoantibodies and with adrenocortical insufficiency, Addison's disease, as one of its cardinal manifestations. Approximately 10-20% of APS I patients suffer from autoimmune hepatitis, which carries a high mortality, if untreated. The presence of putative antigenic targets in the liver was investigated. Cytochrome P4501A2 (CYP1A2) and aromatic L-amino acid decarboxylase (AADC) were identified as hepatic autoantigens with the use of APS I sera for immunofluorescent staining of normal human liver, Western blot of microsomal and cytosol fractions of human liver homogenate, and immunoprecipitation of in vitro transcribed and translated radioactively labelled proteins. The presence of CYP1A2- and AADC-antibodies was significantly correlated to AIH, and CYP1A2 antibodies inhibited enzyme activity in vitro. In conclusion, a daily replacement dose of 50 mg of DHEA sufficiently restores levels of DHEA, DHEA(S) and androgens in women with Addison's disease, without severe side-effects. We have further identified CYP1A2 and AADC as hepatic autoantigens associated with autoimmune hepatitis in APS I.

Las poliaminas regulan procesos de crecimiento y diferenciación celular, y su desregulación está relacionada con diferentes patologías incluyendo el cáncer. Las antizimas (AZs) de ornitina descarboxilasa (ODC) inhiben tanto su biosíntesis, como su captación, regulando los niveles intracelulares de poliaminas. En esta tesis se ha caracterizado una nueva proteína inhibidora de antizimas (AZIN2) que posee alta homología con ODC y el inhibidor de antizimas previamente conocido (AZIN1). Esta nueva proteína está desprovista de actividad enzimática, pero es capaz de revertir la acción que las tres antizimas conocidas ejercen sobre la actividad ODC y la captación de poliaminas. A diferencia de sus proteínas homólogas, AZIN2 se localiza subcelularmente en el ERGIC, y se expresa específicamente en cerebro y testículo, pero de forma muy abundante en espermátidas y espermatozoides, al igual que AZ3, indicando que estas dos proteínas juegan un importante papel regulando los niveles de poliaminas durante la espermiogénesis. / Polyamines regulate cell growth and differentiation, and the alteration of their homeostasis is related to different diseases, including cancer. Ornithine decarboxylase (ODC) antizymes (AZs) regulate polyamine levels by inhibiting both their biosynthesis and the cellular uptake. In this work, a new ODC paralogue has been characterized as a novel antizyme inhibitor protein that has been named AZIN2. This protein lacks decarboxylating activity, but it is able to reverse the action of any of the three antizymes on ODC activity and polyamine uptake. Unlike its homologue proteins ODC and AZIN1, AZIN2 is located in the ERGIC, and it is specifically expressed in brain and testes. The abundant expression in spermatids and spermatozoa, concomitantly with AZ3, suggests that both proteins may play an important role in regulating polyamine levels during spermiogenesis