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The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyses the stereospecific aldol-like condensation between phosphoenolpyruvate (PEP) and the five-carbon sugar D-arabinose 5-phosphate (A5P). This is the first biosynthetic step in the formation of 3-deoxy-D-manno-octulosonate (KDO), an essential lipopolysaccharide component of all Gram-negative bacteria. KDO8PS is evolutionarily related to the shikimate pathway enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), which catalyses a similar condensation reaction between PEP and the four-carbon sugar D-erythrose 4-phosphate (E4P), in the first step of the shikimate pathway to aromatic compounds in plants and microorganisms. As well as being a one-carbon shorter substrate, E4P has the opposite C2-OH configuration to A5P. While there are both metal-dependent and metal-independent forms of KDO8PS, in contrast, all DAH7PS are metal-dependent enzymes. Little is understood about the key sequence features that distinguish KDO8PS and DAH7PS. These features, particularly those that contribute to A5P or E4P binding, are thought to be responsible for the differences in substrate specificity between the two enzymes. This thesis describes the functional and structural studies of KDO8PS mutants to examine the roles of these residues, using the metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and the metal-independent KDO8PS from Neisseria meningitidis. In Chapter 2 an extensive KDO8PS and DAH7PS sequence analysis is presented. The results, which identify sequence conservation in both enzymes, are discussed in the context of the (β/α)8 TIM-barrel structure. Some of the differences in conservation between the two enzymes were highlighted as being obvious in having a role or contributing to the different substrate selection preferences of the two enzymes, such as an extended β7α7 loop in KDO8PS, and motif differences on the β2α2 and β4α4 loops. A similar analysis was also used to compare metal-dependent and metal-independent KDO8PSs, and it was found the two forms differ in the conservation of only three residues. Chapter 3 describes the characterisation of A. ferrooxidans KDO8PS (AfeKDO8PS) and investigates aspects of metal dependency in KDO8PS. The enzyme was found to be metal dependent, and like all other KDO8PS enzymes, to possess a tetrameric quaternary structure, and display tight substrate specificity. The β8α8 loop was found to have a critical role in binding and positioning the substrates, and AfeKDO8PS could not be engineered to be a metal-independent enzyme. The role of the KDO8PS-conserved KANRS motif, present on the β2α2 loop and one of the main contributors to the A5P binding site, is probed in Chapter 4. Individual residues of the motif were mutated to investigate function, and the motif was converted to the equivalent motif found in DAH7PS (KPRS). It was found that the Lys plays a critical role in enzymatic catalysis, and is likely intimately involved in the enzyme mechanism. The Asn residue of the motif in KDO8PS was found to be an important contributor to KDO8PS stereospecificity. The work described in Chapter 5 investigates the role of the β7α7 loop in KDO8PS. This long active-site loop, which exists in a shorter version in DAH7PS, was found not to be essential for catalysis in KDO8PS, but was necessary for efficient catalysis. The two conserved residues on the loop provide interactions to A5P, but the presence of the extended loop as a whole was found to be most important for catalytic efficiency. In Chapter 6 a conserved residue on the re face of PEP is investigated. In KDO8PS the residue is conserved as Asp, and in DAH7PS the same residue is conserved as a Glu. Mutational analysis found that in KDO8PS the Asp residue appears to be important for enzyme activity but unimportant for PEP binding. Mutating this Asp in KDO8PS to Glu was accommodated by KDO8PS, but it was found its introduction could potentially be optimised by coupling the change with mutation to other conserved differences. In KDO8PS, one of the interfaces between adjacent subunits in the tetrameric structure is partially composed of a conserved sequence motif, PAFLxR. In Chapter 7, the roles of the residues in this motif are explored. The Arg of the motif was found to be important for A5P binding. The equivalent (and also conserved) motif in DAH7PS is GARNxQ, and mutation of residues in the KDO8PS motif to the equivalent residues in DAH7PS was tolerated by KDO8PS, but negatively impacted upon the enzyme kinetic parameters. The sequence features investigated in the other chapters were combined with those to the subunit interface to create a DAH7PS-like protein. This extensively engineered protein lost all KDO8PS activity, but nor did it gain DAH7PS activity. Lastly, in Chapter 8 the results from all chapters are reviewed and ideas are discussed for advancing the research presented in this thesis.

The goal of our research is to generate cryopreservation agents derived from antifreeze glycoproteins. One postulated mechanism of cell cryo-injury is ice recrystallization. It is known that simple saccharides and cryopreservation agents (DMSO) display ice recrystallization inhibition (IRI). This study assessed the cytotoxicity and cryopreservation ability of these sugars in relation to their IRI. It was determined that compounds with greater IRI have increased cytotoxicity yet confer cryoprotection. To further investigate how structure is affecting IRI activity, several galactopyranoside derivatives were synthesized. A series of deoxy and α-Callyl- deoxy galactopyranoses were prepared. Testing determined that removal of any hydroxyl group removes IRI. 3-deoxy-β-thiophenyl galactose was also synthesized and had surprisingly better IRI than β-thiophenylgalactose. Also, 6-azido galactose had similar IRI to 6-deoxy galactose. Lastly, a series of β- thioalkylgalactosides was synthesized and testing gave contradicting results which suggest that predicting IRI based on hydrophilicity is more complicated than initially hypothesized.

University, Diss., 2002--Göttingen.

Universiẗat, Diss., 2002--Göttingen.

Made available in DSpace on 2014-06-11T19:22:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-13Bitstream added on 2014-06-13T20:49:39Z : No. of bitstreams: 1 galvao_fc_me_arafcf.pdf: 2113855 bytes, checksum: e2ee31898ce2604cafc3f1e7c7c0c0bd (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / PROPG / O fator de início de tradução 5A (eIF5A) é altamente conservado de arqueas a mamíferos e é essencial para a viabilidade celular. Este fator é a única proteína conhecida que sofre uma modificação pós-traducional única e necessária para a função de eIF5A, em que uma lisina específica é convertida em um resíduo de hipusina pela ação das enzimas desoxi-hipusina sintase (Dys1) e desoxi-hipusina hidroxilase (Lia1). Inicialmente, eIF5A foi relacionada à etapa do início da tradução, porém, dados recentes sugerem a sua atuação na etapa de elongação ao invés de início. No entanto, além do fato de a função específica de eIF5A na célula não ser conhecida, o papel da hipusinação para o funcionamento de eIF5A também não é conhecido. Diante disso, o objetivo deste trabalho é caracterizar mutantes condicionais para o gene da desoxi- hipusina sintase e, dessa forma, contribuir para o entendimento não só da função da hipusinação sobre eIF5A, mas também para o entendimento da função específica de eIF5A na célula. Para isso, foram iniciadas análises de caracterização fenotípica com os alelos dys1Δ1-28 e dys1W75R/T118A/A147T (dys1-1). Inicialmente, foi realizada a subclonagem do alelo dys1Δ1-28 , uma vez que, por ter sido identificado em um rastreamento de duplo-híbrido, este alelo estava em fusão com a região codificadora do domínio de ativação de Gal4. Porém, após realização da subclonagem, ou seja, quando na ausência do domínio de ativação, este alelo não apresentou o fenótipo condicional de crescimento inicialmente observado. Portanto, o mutante se tornou impróprio para a realização dos ensaios subsequentes e foi descartado. Em seguida, foram iniciadas as análises com o alelo dys1-1, nas quais foi observada diminuição nos níveis totais de Dys1 mutada, e consequentemente, diminuição nos níveis de hipusinação. Devido a isso... / The translation initiation factor 5A (eIF5A) is highly conserved from archaea to mammals and is essential for cell viability. This factor is the only known protein that undergoes an unique and essential post-translational modification, in which a specific lysine residue is converted into hypusine by the action of the enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1). Initially, eIF5A was related to the initiation step of translation, however, recent data suggest a role in the elongation step of translation. However, besides the fact that the specific function of eIF5A in the cell is still obscure, the role of hypusination in eIF5A function is unknown. Thus, the goal of this project is to characterize conditional mutants of the deoxyhypusine synthase gene and thereby contribute to the understanding not only the function of hypusination in eIF5A, but also of the specific role of eIF5A in the cell. We started a phenotypic characterization of two different alleles: dys1Δ1-28 and dys1W75R/T118A/A147T (dys1-1). Initially, we performed a subcloning of the allele dys1Δ1-28 , once the allele was fused with the coding region of GAL4 activation domain, due to the fact that this allele is devived of a two-hybrid screening. However, after performing the subcloning, that is, in the absence of the activation domain, this allele showed no conditional growth phenotype as originally observed. Therefore, this mutant has become improper to carry out the subsequent analysis and was discarted. Then, the analyses with dys1-1 allele were initiated, in which it was observed a decrease in total levels of Dys1 and, consequently, a decrease in the hypusination levels. Because of that, this allele shows a decrease in cell growth rate and growth arrests after 24 hours in medium lacking the osmotic regulator. However, this growth arrest is not followed by cell lysis. Furthermore, the mutant ... (Complete abstract click electronic access below)

The goal of our research is to generate cryopreservation agents derived from antifreeze glycoproteins. One postulated mechanism of cell cryo-injury is ice recrystallization. It is known that simple saccharides and cryopreservation agents (DMSO) display ice recrystallization inhibition (IRI). This study assessed the cytotoxicity and cryopreservation ability of these sugars in relation to their IRI. It was determined that compounds with greater IRI have increased cytotoxicity yet confer cryoprotection. To further investigate how structure is affecting IRI activity, several galactopyranoside derivatives were synthesized. A series of deoxy and α-Callyl- deoxy galactopyranoses were prepared. Testing determined that removal of any hydroxyl group removes IRI. 3-deoxy-β-thiophenyl galactose was also synthesized and had surprisingly better IRI than β-thiophenylgalactose. Also, 6-azido galactose had similar IRI to 6-deoxy galactose. Lastly, a series of β- thioalkylgalactosides was synthesized and testing gave contradicting results which suggest that predicting IRI based on hydrophilicity is more complicated than initially hypothesized.

No description available.

No description available.

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase and 3-deoxy-Dmanno- octulosonate 8-phosphate synthase (KDO8P) synthase are two enzymes that catalyse very similar reactions. DAH7P synthase is the first enzyme of the shikimate pathway and catalyses the condensation reaction between the four-carbon sugar erythrose 4-phosphate (E4P) 1 and the three-carbon sugar phosphoenolpyruvate (PEP) 2 to give the seven-carbon sugar DAH7P 3. KDO8P synthase catalyses a similar condensation reaction between the five-carbon sugar arabinose 5-phosphate (A5P) 8 and PEP 2 to give the eight-carbon sugar KDO8P 9. Early mechanistic studies have shown the reaction mechanisms of these two enzymes to be very similar and structural and phylogenic analysis has suggested that the two enzymes share a common ancestor. However, there are differences between the two enzymes that have not been explained by the current literature. Whereas all DAH7P synthases require a divalent metal ion for activity, there exists both metallo and non-metallo KDO8P synthases. As well as this, there is the difference in substrate specificity. The natural substrate of KDO8P synthase, A5P, is one carbon longer and has the opposite C2 stereochemistry to E4P, the natural substrate of DAH7P synthase. This study investigates the role of the C2 and C3 hydroxyl groups of E4P and A5P in the enzyme catalysed reactions. The E4P analogues 2-deoxyE4P 38 and 3-deoxyE4P 39 have been synthesised from [beta]-hydroxy-[gamma]-butyrolactone and malic acid respectively. The two analogues were tested as substrates for DAH7P synthase from a variety of organisms, including N. meningitidis, the purification and characterisation of which was carried out during the course of these studies. It was found that both analogues were substrates for DAH7P synthase. 2-DeoxyE4P was found to be the best alternative substrate for DAH7P synthase to date. The analogous study was carried out on KDO8P synthase from N. meningitidis with 2- deoxyR5P 34 and 3-deoxyA5P 40. It was found that removal of the C2 and C3 hydroxyl groups of A5P was much more catastrophic for the KDO8P synthase catalysed reaction. Commercially available 2-deoxyR5P was found to be a very poor substrate, whereas 3-deoxyA5P, which was prepared according to a literature procedure was not a substrate. The difference in substrate specificities of DAH7P synthase and KDO8P synthase is consistent with the hypothesis that despite their similarities, these two related enzymes have different mechanisms. The key step for DAH7P synthase appears to be coordination of the E4P carbonyl to the divalent metal. The metal appears to play a less important role in the KDO8P synthase reaction and the key step is the correct orientation of A5P in the active site.