The emergence of drug resistant strains of Plasmodium has given a new face to the old disease, malaria. One of the approaches is to block metabolic pathways of the pathogen. The current thesis describes the X-ray crystallographic analysis of two enzymes of the fatty acid biosynthesis pathway of the malaria parasite Plasmodium falciparum. In order to understand the functional mechanism and mode of inhibitor binding, enzyme-inhibitor complexes were characterized, which could help in further improvement of the efficacy of the inhibitors and hence to fight against the disease. The introductory chapter of the thesis presents a discussion on malaria and different metabolic pathways of the pathogen which could be suitable targets for novel antimalarials. In continuation to that, the pathway of our choice the fatty acid biosynthesis and an overview of the structural features of the enzymes involved in the pathway that have been characterized from different organisms are also described. The second chapter includes the tools of X-ray crystallography that were used for structural studies of the present work. It also discusses the biochemical, biophysical and other computational methods used to further characterize the enzymes under study. Triclosan, a well known inhibitor of Enoyl Acyl Carrier Protein Reductase (FabI) from several pathogenic organisms, is a promising lead compound to design effective drugs. The X-ray crystal structures of Plasmodium falciparum FabI (PfFabI), in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations, were determined and compared with triclosan binding which form the basis of chapter 3. The structures revealed that 4 and 2’ substituted compounds have more interactions with the protein, cofactor and solvent molecules as compared to triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2’ position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water based inhibitor design. 2’ and 4’ unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensates for the lost interactions due to the unsubstitution at 2’ and 4’. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2’ and 4’ positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors. Certain residues in the substrate binding tunnel of PfFabI were mutated to identify the role of these residues in substrate binding and protein stability, which forms the 4th chapter of the thesis. The substrate binding site residue Ala372 of PfFabI has been mutated to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli FabI (EcFabI) which has a Methionine at the structurally similar position of Ala372 of PfFabI. Kinetic studies of the mutants of PfFabI and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a 3-fold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. A second set of mutants generated to check this hypothesis exhibited loss of function, except in one case where, the crystal structure showed that the substrate binding loop is stabilized by a water bridge network. The main focus of chapter 5 is β-Hydroxyacyl-acyl carrier protein dehydratase of Plasmoduim falciparum (PfFabZ) which catalyzes the third and important reaction of the fatty acid elongation cycle. The crystal structure of PfFabZ was available in its hexameric (active) and dimeric (inactive) forms. However, until now PfFabZ has not been crystallized with any bound inhibitors. We have designed a new condition to crystallize PfFabZ with its inhibitors bound in the active site, and determined the crystal structures of three of these complexes. This is the first report of the crystal structures of PfFabZ with competitive inhibitor complexes and the first such study on any FabZ enzyme with active site inhibitors. These inhibitors in the active site stabilize the substrate binding loop, revealing the substrate binding tunnel with an overall shape of “U”. In the crystal structure, the residue Phe169 located in the middle of the tunnel was found to be in two different conformations, open and closed, implying that it controls the length of the tunnel and makes it suitable for accommodating longer substrates merely by changing its side chain conformation. The hydrophobic nature of the substrate binding channel signifies the specificity for the hydrophobic tail of fatty acid substrates. The volume of the active site tunnel is determined by the sequence as well as by the conformation of the substrate binding site loop region and varies between organisms for accommodating fatty acids of different chain lengths. All PfFabZ inhibitors reported here bind to the active site through specific contacts like hydrogen bonds with catalytic residues and hydrophobic interactions. This report on the crystal structures of the complexes of PfFabZ provides the structural basis of the inhibitory mechanism of the enzyme, that could be used to improve the potency of inhibitors against an important component of fatty acid synthesis common to many infectious organisms. The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. The analysis discussed in chapter 6, led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects. Rv0098, predicted to be the FabZ of Mycobacterium tuberculosis, was cloned, expressed, purified, crystallized, and X-ray diffraction data were collected. Molecular replacement trials with all “hot dog” fold proteins failed to yield any significant solution due to the low sequence similarity (<20%) of Rv0098 compared to other FabZs. During the trials of structure solution by multiple isomorphous replacement method, structure of Rv0098 was published and it was shown to be a long-chain fatty acyl-CoA thioesterase (FcoT). The crystal structure of Rv0098 did not explain the molecular basis of substrate specificity of varying chain lengths. Molecular dynamics studies were carried out, which revealed that certain residues of the substrate binding tunnel are flexible and thus modulates the length of the tunnel. Flexibility of the loop at the base of the tunnel was also found to be important for determining the length of the tunnel for accommodating appropriate substrates. The structural basis of accommodating long chain substrates by Rv0098 is discussed in chapter 7, by combining the crystallographic and molecular dynamics studies. Part of the work presented in the thesis has been reported in the following publications. Karmodiya, K., Sajad, S., Sinha, S., Maity, K., Suguna, K. and Surolia, N. (2007) Conformational stability and thermodynamic characterization of homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase. IUBMB Life 59, 441-9. Pidugu, L. S., Maity, K., Ramaswamy, K., Surolia, N. and Suguna, K. (2009) Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins. BMC Struct Biol 9, 37. Kapoor, N., Banerjee, T., Babu, P., Maity, K., Surolia, N. and Surolia, A. (2009) Design, development, synthesis, and docking analysis of 2'-substituted triclosan analogs as inhibitors for Plasmodium falciparum enoyl-ACP reductase. IUBMB Life 61, 1083-91. Maity, K., Bhargav, S. P., Sankaran, B., Surolia, N., Surolia, A. and Suguna, K. (2010) X-ray crystallographic analysis of the complexes of enoyl acyl carrier protein reductase of Plasmodium falciparum with triclosan variants to elucidate the importance of different functional groups in enzyme inhibition. IUBMB Life 62, 467-76. Maity, K., Banerjee, T., Narayanappa, P., Surolia, N., Surolia, A. and Suguna, K. (2010) Effect of substrate binding loop mutations on the structure, kinetics and inhibition of Enoyl Acyl Carrier Protein Reductase from Plasmodium falciparum. (Communicated) Maity, K., Bharat, S. V., Kapoor, N., Surolia, N., Surolia, A. and Suguna, K. (2010) Insights into the functional and inhibitory mechanism of the β-Hydroxyacyl-Acyl Carrier Protein Dehydratase of Plasmodium falciparum from the crystal structures of its complexes with active site inhibitors. (Communicated)

Investigations detailed in this thesis constitute a part of the continuing programme of research undertaken in our laboratory on the riboflavin carrier protein (RCP) with partic­ular reference to identification and synthesis of neutralizing antigenic determinants, design of relevant epitope mimetics with improved immunogenic characteristics and relationship between their secondary structures and immunological properties. The riboflavin carrier protein is elaborated as a reproductive stratagem to ensure ade­quate vitamin deposition in the developing oocyte in the chickens. The protein is scrupu­lously conserved through evolution in terms of physico chemical and immunological char­acteristics from fish through birds to mammals, including primates. In rodents and sub­human primates immunization with the heteroantigen viz., chicken egg white RCP leads to functional neutralization of the endogenous maternal protein resulting in curtailment of early pregnancy. Thus, the crucial role of RCP in maintenance of pregnancy is established and the protein identified as a potential candidate vaccine for immimocontraception. Fur­ther studies with the reduced and carboxymethylated (RCM) RCP as the immunogen re­veal that antibodies induced by RCM-RCP are equally effective in bioneutralization of the endogenous protein. So it can be surmised that the native folded structure of RCP is not obligatory for eliciting bioneutralizing antibodies. In an attempt to identify functionally relevant regions of the protein, a panel of monoclonal antibodies (MAbs) have been raised and characterized. One of the MAbs viz., 6J32Ci2 could bring about early fetal resorp-tion when injected to mice with confirmed pregnancies. These results prompted a detail molecular immunological approach to understand underlying mechanisms. The principal aims of the present investigations include: (1) identification of neutralizing epitopes; (2) synthesis of peptidyl sequences incorporating these determinants; (3) an understanding of the structure, antigenic and immunogenic characteristics of these peptides; (4) correlation of conformational and antigenic characteristics; (5) rational design and synthesis of peptide analogs with greater propensity to assume predicted secondary structures; (6) analysis of conformation dependency of peptide antigens and the importance of such conformation in generating an optimal B-cell response; (7) the efficacy of the antibodies elicited by these Peptide antigens in neutralizing endogenous protein with the ultimate aim of designing synthetic vaccines. Chapter 1 of this thesis deals with a general introduction summarizing the current status of knowledge regarding the chemistry and biology of RCP as well as synthetic pep­tides as potential immunogens. Chapter 2 outlines details of the experimental procedures adopted. Chapter 3 describes the results of investigations on the C-terminal fragment (residues 200-219) of cRCR The main consideration in selecting this sequence for the design of a potential peptide-based vaccine relied on the epitopic specificity of the neu­tralizing MAb 6S2C12. Epitope mapping using the Pepscan method revealed that the monoclonal antibody recognizes a core sequence corresponding to residues 203-210 of the cRCP. A 21-residue synthetic peptide (C-21) comprising this epitope was synthesized and antibodies elicited to the peptide conjugated to two different carriers, namely diphtheria toxoid and purified protein derivative (PPD) for T-cell help. In both active and passive immunoneutralization experiments, the peptide specific neutralizing antibodies interfered with the biological function of the protein and hence either protected from pregnancy or caused early fetal resorption in rodents as well as in sub-human primates. The conforma-tional properties of the peptide in aqueous buffers were analyzed from circular dichroism which revealed the absence of any ordered structure in the native C-21 peptide. Theoreti­cal predictions of secondary structure suggested a propensity for an t*-helical structure for this fragment in the native protein. Therefore, influence of the helix-promoting solvent, vizM 2,2,2,trifluroethanol (TFE) on the C-21 peptide was investigated. Addition of TFE resulted in spectral changes with negative bands at 208 and 222 nm and a positive band at 190 rim which are typical of an a-helix. To gain more information on the conformational characteristics of this peptide, it was considered worthwhile to stabilize the native peptide in an a-helical conformation based on simple rational design principles. Towards this end, four analogs of the parent peptide were synthesized and helix stabilization was sought to be achieved by introducing either salt bridges or back-bone conformational constraints such as by incorporating a-amino isobutyric acid at appropriate positions. In all the analogs, the core sequence, recognised by the neutralizing MAb 6B2C12 was maintained intact to ensure induction of antibodies capable of recognizing the native protein. CD spectral analysis of the analog peptides indicated that all the engineered peptides had varying degrees of enhanced helicities as compared to the parent peptide. The immunogenicity of each analog was studied by to the relevant peptide-diphtheria toxoid conjugates and analyzing their reactivities with the native protein by direct and competitive ELISA. The results revealed that these engi­neered conformational analogs axe highly immunogenic eliciting high titers of anti-protein antibodies. The relative affinities of these antibodies to bind cRCP were investigated. The antibodies to peptide analogs had higher affinities for the native protein and a positive correlation was found between the helical content of the peptide antigen in question and the relative affinity of corresponding antibody. The antibodies directed to all the peptide analogs could block the function of RCP resulting in early embryonic resorption when ad­ministered to pregnant mice. An interesting pattern of immunological cross-reactivity has been observed with the native and designed peptides. Antibodies raised to constrained helical analogs could bind the C-21 peptide which is structurally flexible. In contrast, the antibodies raised to the flexible native peptide antigen were inefficient in recognizing the structured peptides. The ability of all the peptide antibody to bind the native protein has been interpreted in terms of a conformationally flexible C-terminus region in cRCP. Chapter 4 details investigations on a 21-residue peptide (N- 21) from the N-terminiis (4-24) of the protein. Selection of this peptidyl sequence relied on theoretical prediction of potential sequential determinants on RCP other than at C-terminus as well as on the outcome of immunoneutralisation experiments using antibodies to egg yolk RCP which lacks the relevant C-terminal determinants. The structure of this peptide in solution was analyzed by two dimensional NMR and CD. NMR experiments revealed the presence of two structured regions in the peptide. Diagnostic nuclear Overhauser effects characteristics of reverse turns or short frayed helical segments over residues 3-9 and 18-21 of the peptide were obtained. CD spectra showed the presence of a strong, negative band at 204 nm over a wide range of solvent conditions, a feature which has been interpreted in terms of a "polyproline Il-like" segment encompassing residues 11-16 which corresponds to an interesting (X-Pro)^ repeat in the N-21 sequence. Specific antibodies were generated to this peptide as a conjugate with diphtheria tox­oid. Administration of the antipeptide antibodies could neutralize the protein in vivo as demonstrated by early embryonic loss in pregnant mice. In limited experiments the anti­peptide antibodies showed propensity to protect bonnet monkeys from pregnancy over a few consecutive ovulatory cycles when titres are maintained elevated by periodic boosting. To address the relationship between peptide structures and antigenicity, epitope mapping of this antipeptide antibodies as well- as the polyclonal antibodies to native RCP was undertaken using the Pepscan method. The results reveal that antigenic regions correspond well to conformationally well-defined elements of structure with the polyproline II-like seg­ment being a common antigenic determinant on both the peptide and the native protein. These observations are suggestive of the involvement of both the N and C-terminal regions of RCP in terms of its binding to putative plasma membrane receptors.

J'étudie la formation du système vasculaire des feuilles des plantes à l'aide de modèles mathématiques. L'hypothèse de canalisation d'une phytohormone, l'auxin, stipule que l'auto activation de son transport entre les cellules crée des chemins préférentiels qui se différencieront plus tard en système vasculaire. J'entreprends une analyse numérique de modèles de canalisation sur une grande matrice et parviens à créer des motifs branchés dans lesquels circule l'auxin. Une analyse de stabilité d'un modèle simplifié nous éclaire sur les raisons de la formation de ces motifs et l'impossibilité de créer un réseau réticulé. La majorité des plantes ayant un système vasculaire réticulé, je modifie le modèle de façon à obtenir ce type de réseau. En ajoutant une variable biologiquement plausible je parviens à créer un réseau réticulé dans lequel l'auxin circule uniformément. Enfin, je discute des relations entre la formation du système vasculaire et de la spirale de phyllotaxie.

ACP interacts with diverse proteins in an unknown way. Possibly there is a similar mode of interaction between ACP and all ACP-binding proteins, the amphiphilic helix. The hydrophobicities of helices from 4 different ACPs were compared. Hydrophobic moment plots were prepared for ACP helices and those of many EF hand calcium-binding proteins. Both groups of proteins occupied the same region of the plot.

Many bacteria use inorganic nitrogen and sulfur compounds for energy metabolism. These compounds are often toxic and so bacteria must adapt to survive their deleterious effects. Bacteria use specific proteins in order to metabolise, sense and detoxify these compounds. In this thesis protein interactions with inorganic nitrogen and sulfur compounds are examined at the mechanistic level. Intermediates in the Sox sulfur oxidation pathway are covalently attached to a cysteine on the swinging arm of the substrate carrier protein SoxYZ. An interaction between the Sox pathway enzyme SoxB and the carrier protein SoxYZ is demonstrated. A crystal structure of a trapped SoxB-SoxYZ complex at 3.3 &Aring; resolution identifies two sites of interaction, one between the SoxYZ carrier arm and the SoxB active site channel and the other at a patch distal to the active site. The presence of a distal interaction site suggests a mechanism for promiscuous specificity in the protein-protein interactions of the Sox pathway. Using biophysical methods it is shown that SoxB distinguishes between the substrate and product forms of the carrier protein through differences in interaction kinetics and that the carrier arm-bound substrate group is able to out-compete the adjacent C-terminal carboxylate for binding to the SoxB active site. The thiosulfate dehydrogenase TsdA has an unusual His/Cys coordinated heme. TsdA catalyses oxidative conjugation of two thiosulfate molecules to form tetrathionate. Mass spectrometry and UV/visible spectroscopy are used to identify an S-thiosulfonate reaction intermediate which is covalently attached to the cysteine heme ligand. A catalytic mechanism for TsdA is proposed using a crystal structure of TsdA at 1.3 &Aring; resolution alongside site-directed mutagenesis of active site residues. Nitric oxide is produced by the mammalian immune response to kill bacterial pathogens. Part of the killing mechanism occurs through the reaction of nitric oxide with protein-bound iron-sulfur clusters. However, the same type of reaction is also exploited by nitric oxide-sensing bacterial proteins. An infrared spectroscopy approach is developed to detect the products of iron-sulfur protein nitrosylation. Using this methodology it is shown that the presence of trace O2 strongly impacts which products are formed in these nitrosylation reactions. These observations are of physiological relevance because bacteria are often exposed to NO under aerobic conditions during an immune response.

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2016-10-03T18:49:30Z No. of bitstreams: 1 daniellabalduinovictorino_dissert.pdf: 2037812 bytes, checksum: ec4892555795968e4470a593baca8ebc (MD5) / Made available in DSpace on 2016-10-03T18:49:30Z (GMT). No. of bitstreams: 1 daniellabalduinovictorino_dissert.pdf: 2037812 bytes, checksum: ec4892555795968e4470a593baca8ebc (MD5) Previous issue date: 2014-10-06 / Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP / Introduction Down syndrome (DS) is caused by the presence of three copies of chromosome 21 in consequence to chromosome nondisjunction in maternal meiosis observed in about 95% of cases. Genetic polymorphisms involved in folate metabolism were associated with the maternal risk for DS. However, the results are contradictories. Objectives To perform a systematic review and meta-analysis in order to evaluate the association between Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, Methionine synthase reductase (MTRR) A66G, Methionine synthase (MTR) A2756G, Reduced folate carrier 1 (RFC1) A80G, Cystathionine β-synthase (CβS) 844ins68, Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) G1958A and Transcobalamin 2 (TC2) C776G genetic polymorphisms and the maternal risk for DS. Methods Studies were searched up to May 2014 on MEDLINE, EMBASE, LILACS, hand searched reference lists of published articles and conference meetings and personal communication. Case-control studies that evaluated the association between genetic polymorphisms in case mothers (DSM) and controls mothers (CM) were included. DSM are considered mothers that have gave birth to children with free trisomy of 21 chromosome and CM are considered mothers that have gave birth to children without chromosomal abnormality, syndrome or malformation. Studies with mothers of DS individuals with translocation or mosaicism, case reports, editorials and review articles were excluded. Data extraction and quality assessment were performed independently by two investigators. Meta-analysis assesses the associations between each genetic polymorphism and maternal risk for DS by dominant, recessive, codominant and allelic genetic models. Dichotomous outcome measures were pooled using fixed and random effects models and the results were expressed by odds ratio (OR) with 95% confidence intervals (95% CI). Heterogeneity between studies was evaluated using Q test and the I2 and subgroup and sensitivity analyses were performed in order to investigate the potential sources of heterogeneity. Publication bias was estimated using funnel plot and linear regression test. Results Collectively, 30 case-control studies including 3,101 DSM and 3,967 CM were included. Significant association between MTHFR C677T and MTRR A66G polymorphisms and maternal risk for DS was found when all population is considered. Subgroup and sensitivity analyses according ethnicity showed significant associations for the MTHFR C677T polymorphism in Caucasians, Brazilians and Asians and for the MTRR A66G polymorphism in Caucasians. Additionally, the results of the RFC1 A80G polymorphism demonstrated significant association, it was also found in Asians and maternal age less than 35 years at conception subgroups analyses. Finally, MTHFD1 1958GA genotype was revealed as maternal risk factor for DS when only studies with control group in Hardy-Weinberg equilibrium were considered. No association among MTHFR A1298C, MTR A2756G, CβS 844ins68 and TC2 C776G polymorphisms and maternal risk for DS was found. Conclusions MTHFR C677T, MTRR A66G, RFC1 A80G and MTHFD1 1958GA polymorphisms are associated with maternal risk for DS. / Introdução Síndrome de Down (SD) é atribuída à presença de três cópias do cromossomo 21, decorrente da não-disjunção cromossômica meiótica materna em 95% dos casos. Polimorfismos genéticos maternos envolvidos no metabolismo do folato foram associados ao nascimento de indivíduos com a SD, porém os resultados dos estudos são contraditórios. Objetivos Avaliar, por meio de revisão sistemática e metanálise, a associação entre os polimorfismos genéticos maternos Metilenotetrahidrofolato redutase (MTHFR) C677T e A1298C, Metionina sintase redutase (MTRR) A66G, Metionina sintase (MTR) A2756G, Carreador de folato reduzido 1 (RFC1) A80G, Cistationina β-sintase (CβS) 844ins68, Metilenotetrahidrofolato desidrogenase 1 (MTHFD1) G1958A e Transcobalamina 2 (TC2) C776G e o nascimento de indivíduos com a SD. Métodos As buscas bibliográficas foram realizadas anteriormente a maio de 2014 e os bancos de dados utilizados foram: PUBMED, EMBASE, LILACS, lista de referências bibliográficas dos artigos selecionados, busca manual em anais de congressos e comunicação pessoal. Foram incluídos estudos caso-controle que avaliaram a presença dos polimorfismos genéticos em mães de crianças com SD por trissomia livre do cromossomo 21 (mães-caso) e em mães de crianças sem histórico de anormalidades cromossômicas, síndromes ou malformações (mães-controle). Os critérios de exclusão consistiram em estudos que incluíram mães de crianças com SD por translocação ou mosaicismo, relatos de caso, editoriais e artigos de revisão. A extração dos dados e a avaliação da qualidade dos estudos foram feitas por dois investigadores. A metanálise avaliou a associação entre cada polimorfismo e o risco materno para a SD por meio dos modelos genéticos dominante, recessivo, codominante e alélico. Medidas de desfecho dicotômicas foram sumarizadas utilizando-se modelos de efeito fixo e randômico e os resultados foram expressos em odds ratio (OR) com intervalo de confiança de 95% (IC 95%). A heterogeneidade entre estudos foi calculada pelo teste Q e pela estatística I2 e suas potenciais fontes foram investigadas pelas análises de sensibilidade e subgrupo. O viés de publicação foi estimado pelos funnel plot e teste de regressão linear. Resultados Coletivamente, 30 estudos caso-controle preencheram os critérios de elegibilidade, o que totalizou 3.101 mães-caso e 3.967 mães-controle. Foi verificada associação significativa entre os polimorfismos MTHFR C677T e MTRR A66G e o risco materno para SD. As análises de subgrupo de acordo com a etnia revelaram associações significativas para o polimorfismo MTHFR C677T e o risco materno para a SD em caucasianos, brasileiros e asiáticos e para o polimorfismo MTRR A66G em caucasianos. Adicionalmente, foi encontrada associação significativa para o polimorfismo RFC1 A80G e o risco materno para a SD e também nas análises de subgrupo de asiáticos e de mães com idade materna inferior a 35 anos no momento da concepção. Finalmente, o genótipo MTHFD1 1958GA revelou-se fator de risco materno para o nascimento de indivíduos com SD quando a análise foi restringida aos estudos cujo grupo controle estava em equilíbrio de Hardy-Weinberg. Nenhuma associação foi verificada para os polimorfismos MTHFR A1298C, MTR A2756G, CβS 844ins68 e TC2 C776G. Conclusões Os polimorfismos MTHFR C677T, MTRR A66G, RFC1 A80G e MTHFD1 G1958A são fatores de risco materno para a SD.

This thesis entitled ‘ Unusual Acylation Properties of Type II Fatty Acid Biosynthesis Acyl Carrier Proteins’ describes the discovery of self-acylation and malonyl transferase activity in acyl carrier proteins involved in type II fatty acid biosynthesis and assigns a physiological role to these processes inside the cellular milieu. Acyl carrier protein (ACP) is one of the most abundant proteins present inside the cell and almost 4% enzymes require it as a cofactor. Acyl carrier proteins can exist either as discrete proteins or as domains of large functional proteins. They function in a variety of synthases as central molecules to which growing acyl intermediates and nascent product molecules are covalently tethered during the elongation and modification steps required to produce the final product. A prototypical bacterial ACP is composed of 70-80 amino acids and is generally expressed in the apo form. It is post-translationally modified to active holo form by the addition of 4'-phosphopantetheine moiety to an absolutely conserved serine residue in a reaction catalyzed by holo-ACP synthase or 4'-phosphopantetheine transferase. Chapter 1 surveys literature related to carrier proteins inside the cell and describes the thesis objective. It also presents an overview of the acyl carrier proteins and their involvement in various metabolic pathways inside the cell. The chapter details the structural organization of acyl carrier proteins from various sources revealing the conservation in their structure and also details the molecular basis of interaction of ACP with other enzymes inside the cell. The discovery of unusual self-acylation property in acyl carrier proteins involved in polyketide biosynthesis and its absence in acyl carrier proteins involved in fatty acid biosynthesis prompted me to investigate the reasons for this selective behavior. Discovery of self-acylation property in acyl carrier proteins Plasmodium falciparum and chloroplast targeted Brassica napus acyl carrier proteins involved in type II fatty acid biosynthesis and the mechanism of this reaction forms the basis of Chapter 2. In this chapter it has been shown that self-acylation property is intrinsic to a given acyl carrier protein and is not dependent on the pathway in which it is involved. Based on primary sequence analysis and site directed mutagenesis studies presence of an aspartate/glutamate has been identified to be critical for the self-acylation event. Furthermore, it has also been shown that the self-acylation event in type II fatty acid biosynthesis acyl carrier proteins is highly specific in nature employing only dicarboxylic acid –CoAs as substrates unlike the polyketide biosynthesis acyl carrier proteins which utilize both dicarboxylic acid and β-keto acid thiol ester -CoAs as substrates. The detailed kinetics of these reactions has also been worked out. Combining all the results a plausible mechanism for the self-acylation reaction has been proposed. Chapter 3 describes the discovery of a novel malonyl transferase behavior in acyl carrier proteins involved in type II fatty acid biosynthesis. Malonyl transferase property in ACPs of type II FAS from a bacterium (Escherichia coli), a plant (Brassica napus) and a parasitic protozoon (Plasmodium falciparum) were investigated to present a unifying paradigm for the mechanism of malonyl transferase behavior in ACPs. Identification of malonyl transferase property in Plasmodium falciparum ACP and Escherichia coli ACP (EcACP) and the absence of this property in Brassica napus ACP has been described in this chapter. Detailed investigations demonstrated that presence of an arginine or a lysine in loop II and an arginine or glutamine at the start of helix III as the residues that are critical for the transferase activity. In order to assign a physiologic function to these unusual acylation properties, fabD(Ts) mutant strain of Escherichia coli was utilized for heterologous complementation by the various wild type and mutant ACPs that are able to catalyze either or both of the activities. Growth of the mutant strain at non-permissive temperature, when complemented with ACPs catalyzing both the reactions confirmed that these properties have a physiologic relevance. Extensive mutagenesis experiments in conjunction with complementation studies allowed me to propose a plausible mechanism on how the self-malonylation and malonyl transferase properties operate in tandem. Chapter 4 describes the thermodynamic characterization of self-acylation process using Isothermal Titration Calorimetry. Isothermal Titration Calorimetric studies on the binding of malonyl, succinyl, butyryl and methylmalonyl –CoA to Plasmodium falciparum and Brassica napus acyl carrier proteins were performed to investigate the role of thermodynamic parameters in the specificity of self-acylation reaction. Calculation of the parameters showed that the thermodynamics does not control the self-acylation reaction. The evolution of self-acylation property in various acyl carrier proteins and its possible significance in the evolution of various metabolic events is described in Chapter 5. Extensive bioinformatics search was performed and phylogenetic analysis on acyl carrier proteins from 60 different taxa was done using the MEGA4 program. Analysis showed that this property was first found in cyanobacterium. Later, during the course of evolution this property was lost in most acyl carrier proteins, and was retained either in acyl carrier proteins that are targeted to organelles of cyanobaterial orgin viz. apicoplast in apicomplexans and chlorplasts in plants or in acyl carrier proteins involved in secondary metabolic events such as polyketide biosynthesis. Chapter 6 summarizes the findings of the thesis. Acyl carrier protein from Plasmodium falciparum, Brassica napus and Escherichia coli were characterized for their self-acylation and malonyl transferase properties and a combined mechanism for these two properties is proposed. The work done also provides an in vivo rationale to these in vitro processes. Furthermore, the evolutionary significance of the self-acylation behavior is also discussed in the thesis. The thesis also probes into the thermodynamics of the self-acylation reaction in Plasmodium falciparum and Brassica napus acyl carrier proteins. Thus, the thesis adds a new dimension to the much unexplored ACP biology and paves the way to study in vivo roles of these processes in detail. Appendix I describes the Isothermal Titration calorimetric characterization of binding of various acyl-PO4 molecules to Escherichia coli PlsX (Acyl-phosphate acyltransferase). PlsX, the first enzyme of phosphatidic acid biosynthesis pathway catalyzes the conversion of acyl-ACP into acyl-PO4, which is further used by other enzymes leading to the formation of phosphatidic acid. ITC results presented in this section show that longer chain length acyl-PO4 molecules show better binding to PlsX, as compared to the smaller ones demonstrating that long chain acyl molecules serve as better substrates for phosphatidic acid synthesis.

Les vaccins conjugués furent développés suite à l’inefficacité des vaccins polysaccharidiques chez les nourrissons et les personnes âgées. Les vaccins conjugués sont composés d’un polysaccharide extrait de la capsule bactérienne et d’une protéine porteuse. Celle-ci permet de décupler la réponse immunitaire, permettant aux vaccins d’être efficaces. L’évolution des connaissances en chimie et en analytique permettent aujourd’hui de mieux caractériser ces vaccins et de mieux maîtriser leur production. Cependant, les chimies de conjugaison utilisées pour lier le polysaccharide et la protéine porteuse, ne sont pas toujours définies et cela mène souvent à l’obtention de produits hétérogènes. Les objectifs de cette thèse ont été d’étudier le polysaccharide, les protéines porteuses et de nouvelles voies de conjugaisons pour lier spécifiquement ces deux biomolécules.Différents outils analytiques ont été utilisés afin d’acquérir une meilleure connaissance des deux partenaires de conjugaison. Cela a également permis d’établir une stratégie d’analyse efficace pour caractériser les produits de réaction. La spécificité des réactions de conjugaison a été induite par l’utilisation d’espaceurs bi-fonctionnels, réagissant spécifiquement sur certains acides aminés. Leur réactivité a d’abord été testée sur un modèle peptidique. Cela a permis de faciliter la caractérisation et d’étudier l’efficacité et la spécificité des réactions. Les réactions efficaces ont ensuite été testées différents modèles : de la protéine au vaccin. Sur les quatre réactions testées, une a été efficace sur tous les modèles. Cette chimie de conjugaison est prometteuse pour le développement de nouveaux vaccins / Conjugate vaccines were developed because polysaccharide vaccines were not efficient in infant and old people. These vaccines were composed of the polysaccharide extracted from the bacterial capsule linked to a carrier protein. This protein created an immunological boost which allowed the vaccine to induce a proper protection for everyone. As chemistry knowledge and analytical techniques evolved, vaccines can now be better characterized and the production can be better controlled. Nevertheless, the chemistries used to bind the polysaccharide and the carrier protein are not always well-defined, which leads to the production of heterogeneous products. The objectives of this PhD were to study the polysaccharide, carrier proteins and new conjugation chemistries to specifically bind the two biomolecules. The other challenge was to be able to check the reaction specificity and characterize reaction products.To do so different analytical tools were used to allow a better knowledge of both conjugation partners but also to establish an efficient analytical strategy for glycoconjugate characterization. Conjugation reactions specificity was induced by using different bi-functional linkers, reacting specifically for one type of amino acid. Linkers’ reactivity was first tested on a model peptide. This allowed to facilitate the characterization and to check for both reaction specificity and reaction success. Efficient reactions were then tested on different models from carrier proteins to glycoconjugate vaccines. One of the four tested reactions was efficient from the peptide to the vaccine model. This conjugation is thus promising for the development of new conjugate vaccines

No description available.

Malaria, caused by Plasmodium, is one of the most devastating infectious diseases of the world in terms of mortality as well as morbidity (WHO, 2002). The development of resistance in the Plasmodium falciparum against the present antimalarials has made the situation very alarming (Trape et al., 2000). To combat this situation, new antimalarials as well as identification of new drug targets are urgently required. The discovery of the presence of type II fatty acid biosynthesis system in the malarial parasite has offered several promising new targets for this mission. This thesis describes the successful cloning of fabZ from Plasmodium falciparum, its expression in E. coli, single step affinity purification, kinetic characterization and most importantly discovery of two small molecule inhibitors (Sharma et al., 2003). The study was executed to gain insights into the structure and function of PfFabZ to get better understanding of the interactions with its substrate analogs, novel inhibitors and also acyl carrier protein (PfACP). The molecular details of the interactions of the two novel inhibitors were also determined. Lastly, the residues of PfFabZ important for the interaction with PfACP were successfully elucidated. Chapter 1 presents a brief review of the literature about the disease as well as the life cycle, biology and the metabolic pathways operational in malarial parasite, Plasmodium falciaparum. The discovery of type II FAS in P. falciparum and the aims and the scope of the thesis are also discussed. The quest of developing new antimalarials, study of the mechanism of actions of antimalarials such as quinine and its derivatives along with the major metabolic pathways (Purine, pyrimidine, phospholipids, carbohydrate metabolism, folate and heme biosynthesis pathways etc.) existing in P. falciparum are described in detail in this chapter. Origin and importance of apicoplast in P. falciaprum is also described in brief. For long, it was believed that Plasmodium spp. are incapable of de novo fatty acid synthesis but this view has undergone substantial revision due to the recent discovery of plant and bacterial type of fatty acid biosynthesis pathway in them (Surolia and Surolia, 2001). As this pathway is distinct from that of the human host it has accelerated the momentum for the discovery of new antimalarials (Surolia and Surolia, 2001). The Chapter also surveys the details of type II FAS in bacteria, particularly that of E. coli (Rock and Cronan, 1996). The dehydratase step which is the third step of fatty acid elongation cycle has been covered in considerable detail. Lastly, it focuses on the recent advancement in the understanding of fatty acid biosynthesis system in Plasmodium falciparum along with some inhibitors targeting the malarial FAS. As each enzyme of the Plasmodium FAS can serve as good antimalarial targets, my work focuses on the dehydratase step catalyzed by β-hydroxyacyl-ACP dehydratase (PfFabZ). Cloning, expression and kinetic characterization of PfFabZ forms the major content of Chapter 2. The PlasmoDB data base was searched for this gene and the mined out open reading frame contained sequence of the putative FabZ together with the bipartite leader polypeptide. Our aim was to clone the mature PfFabZ without the bipartite leader sequence. Amplification of the mature pffabZ using Plasmodium falciparum genomic DNA revealed the presence of an intron in the ORF and the gene was finally cloned by RT-PCR in pET-28a(+) vector. It was expressed with an N-terminal hexahistidine tag in BL-21(DE3) cells and purified to near homogeneity but the protein was insoluble and unstable. Truncation of 12 residues from the N-terminal end improved the stability and solubility of the protein by 3-5 fold. Truncated PfFabZ was used for all future experiments. FabZs from other sources are reported to be hexamer in solution but PfFabZ showed homodimeric arrangement in the conditions used for gel filtration as well as dynamic light scattering studies. Kinetics of PfFabZ was characterized using substrate analogs, β-hydroxybutryl-CoA (forward substrate) and Crotonoyl-CoA (reverse substrate). Both the forward and reverse reaction were thoroughly characterized by spectrophotometry and HPLC and the reverse reaction was found to be 7 times faster than the forward reaction. Km οf crotonoyl-CoA was calculated to be 86 µM and kcat/Km of 220 M-1s-1 whereas the Kmfor β-hydroxybutryl-CoA was found to be 199 µM and kcat/Kmof 80.2 M-1s-1. The kinetic data clearly indicates the higher affinity of PfFabZ for the reverse substrate. Chapter 3 describes the discovery of two small molecules inhibitors, NAS-21 and NAS-91 for PfFabZ, their detailed inhibition kinetics and their effect on the growth of Plasmodium falciparum in culture. These inhibitors were the first inhibitors to be reported for FabZ class of enzymes with an IC50 ranging below 15 µM. Both of them inhibited PfFabZ following competitive kinetics with respect to the substrates utilized for both the forward and reverse reactions. The inhibition data were analyzed by Lineweaver-Burk and Dixon plots and both inhibitors showed competitive inhibition kinetics with dissociation constant in submicromolar range. Binding constants for both the inhibitors were also determined by fluorescence titration method and were calculated to be 1.6 (± 0.04) X 106 M-1 for NAS-91 and 1.2 (± 0.03) X 106 M-1 for NAS-21. These inhibitors were checked on Plasmodium falciparum culture and both inhibited parasite growth with IC50 values of 7 µM and 100 µM for NAS-21 and NAS-91, respectively. They also inhibited the incorporation of [1,2-14C]-acetate in the fatty acids of the P. falciparum conforming the inhibition of fatty acid biosynthesis. FabZ class of enzymes are thought to contain His-Glu as a catalytic dyad. Based on the disparity in the arrangement of residues at the active site of the dimeric (Swarnamukhi et al., 2006) and hexameric forms of PfFabZ in the crystal structures (Kosteriva et al., 2005), we set out to elucidate the active site residues in PfFabZ which is described in Chapter 4. The role of each of the presumed active site residues His-133 and Glu-147 along with Arg-99 and His-98 were analyzed by chemical modification studies and site directed mutagenesis. Single and double mutants were prepared and the activity of the mutants was monitored by spectrophotometry and isothermal titration calorimetry (ITC). It was concluded that in PfFabZ, His-133 and Glu-147 makes the catalytic dyad, His-98 might be important in directing the substrate in correct orientation while Arg-99 is involved in maintaining the active site loop in proper orientation rather than taking direct part in catalysis. Chapter 4 also concludes that dimeric form of PfFabZ is inactive species and turns into active hexameric form in the presence of substrate. Chapter 5 describes the molecular details of NAS-21 and NAS-91 interactions with PfFabZ. The fact that both these compounds inhibited PfFabZ in competitive manner, prompted me to examine their interaction with the residues in the active site tunnel. Apart from the His-133 and Glu-147 catalytic dyad the only polar residue is His-98 and chemical modification and site directed mutagenesis studies were done to elucidate the interactions of these residues with NAS-21 and NAS-91. Both the inhibitors were able to protect the modification of histidines by DEPC in wild type PfFabZ, His-98-Ala mutant and His-133-Ala mutant but with differential strength, indicating that they do interact with histidines. The interaction of these inhibitors was further confirmed by determining the dissociation constants of wPfFabZ, His-98-Ala, His-133-Ala, His-98-Ala/His-133-Ala double mutant, Glu-147-Ala mutant by fluorescence titration method. The results obtained from chemical modification and fluorescence titration studies confirmed that NAS-21 interacts strongly with histidines, His-98 and His-133 but not with Glu-147. On the other hand NAS-91 interacts loosely with His-98 and His-133 but strongly with Glu-147. Chapter 5 concludes with the observation that both the inhibitors (NAS-21 and NAS-91) interact with the active site residues of PfFabZ, preventing the substrate to enter the active site tunnel. Acyl carrier protein (ACP) is a small acidic protein to which the acyl chain intermediates are tethered and shuttled from one enzyme to another for the completion of fatty acid elongation cycle. Whenever acyl carrier proteins are expressed in E. coli, they are present in three forms apo, holo and acyl-ACPs. Chapter 6 describes a novel method for the expression of histidine tagged PfACP in pure holo form, protocol for the cleavage of his-tag from PfACP by thrombin preparation of homogenous singly enriched ie PfACP [15N]-labeled or [13C]-labeled PfACP as well as doubly enriched [15N]-[13C] PfACP samples for its structure elucidation by NMR (Sharma et al., 2005). These studies also constituted reporting of a holo-ACP structure from any of the sources for the first time (Sharma, et. al. 2006). The purified pure holo-PfACP was further used for the interaction studies with PfFabZ. Earlier studies have shown that ACP interacts with FAS enzymes via helix II with conserved set of residues but the molecular details of the interactions are poorly known (Zhang, et. al., 2003). We have recently solved the NMR structure (Sharma, et. al., 2006) of PfACP and crystal structure of PfFabZ (Swarnamukhi, et. al., 2006). So, both the structures were docked using Cluspro server. Chapter 7 elucidates the roles of important residues on PfFabZ surface near the active site entry which are responsible for interacting with PfACP. The residues lining the active site entry were identified and mutated. The residues lining the active site tunnel of PfFabZ are Arg102, Lys104, Lys105, Lys123, Leu94, Phe95, Ala96, Gly97, Ile128, Ile145, Phe150 and Ala151. Charged residues were mutated to alanine and also to oppositely charged residues while the neutral residues were changed to charged residues. The interaction of PfFabZ mutants with PfACP was studied by ACP independent enzymatic assay and surface plasmon resonance (SPR) spectroscopy. It was concluded that PfFabZ and PfACP interaction is mainly governed by electrostatic interaction made by the charged residues (Lys104 being the most important residue) and is fine tuned by hydrophobic interactions. Chapter 8 summarizes the findings of the thesis. FabZ from Plasmodium falciparum was cloned and biochemically characterized. Two inhibitors for this enzyme were discovered and their molecular details of binding to PfFabZ were elucidated. The presence of catalytic dyad was confirmed and finally the residues of PfFabZ important for interaction with PfACP were elucidated. Appendix I describes the inhibition of PfENR (enoyl ACP reductase), the rate limiting and the fourth enzyme of the fatty acid elongation pathway by green tea extracts. Three tea catechins (EGCG, EGC and ECG) and two plant polyphenols (quercetin and buteine) were selected for the inhibition study. All the catechins inhibited PfENR potently with Ki values in nanomolar range. Among the five compounds studied, EGCG was found to be the best inhibitor. All of them blocked the NADH binding site showing competitive kinetics with respect to NADH and uncompetitive kinetics with crotonoyl-CoA, the substrate analog. Most importantly, the catechins potentiated the inhibition of PfENR by triclosan, a well known PfENR inhibitor. We also report that in the presence of tea catechins triclosan behaves as a slow-tight binding inhibitor of PfENR. The overall inhibition constant of triclosan in the presence of EGCG was calculated to be 2pM which is 50 times better than the earlier reported values with NAD+ (Kapoor, et. al., 2004).