Blastocystis is an important parasite that infects humans and a wide range of animals like rats, birds, reptiles, etc. infecting a sum of 60% of world population. It belongs to the Stramenopiles, a Heterologous group that includes for example the Phythophthora infestans the responsible for the Irish potato famine. Previous work had reported the presence of an unusual fusion protein that is composed of two of the main glycolytic enzymes; Triosephosphate isomerase-glyceraldehyde-3-phosphate dehydrogenase (TPI-GAPDH). Little is known about this protein. Blastocystis TPI-GAPDH and Blastocystis enolase were both characterized biochemically and biophysically in this project. The phylogenetic relationships of those two proteins among other members of either Stramenopiles, or other members of the kingdom of life were examined and found to be grouping within the chromalveolates. Our studies revealed that those two proteins, Blastocystis enolase and Blastocystis TPI-GAPDH, had a peptide signal targeting them to the mitochondria. This was an unusual finding knowing that text books always referred to the glycolytic pathway as a canonical cytoplasmic pathway. Structural studies had also been conducted to unravel the unknown structure of the fusion protein Blastocystis TPI-GAPDH. X-ray crystallography had been conducted to solve the protein structure and the protein was found to be a tetrameric protein composed of a central tetrameric GAPDH protein flanked with two dimmers of TPI protein. Solving its structure would be the starting point towards reviling the role that TPI-GAPDH might play in Blastocystis and other organisms that it was found in as well. Although a fusion protein, the individual components of the fusion were found to contain all features deemed essential for function for TPI and GAPDH and contain all expected protein motifs for these enzymes.

Abstract The aim of this PhD-study was to address some of the overlapping bottlenecks in protein engineering and metagenomics by developing or applying new tools which are useful for both disciplines. Two enzymes were studied as an example: Triosephosphate Isomerase (TIM) and Uridine Phosphorylase (UP). TIM is an important enzyme of the glycolysis pathway and has been investigated via means of protein engineering, while UP is a key enzyme in the pyrimidine-salvage pathway. In this thesis TIM was used to address protein engineering aspects, while UP was used in regards to some metagenomic and bioinformatic aspects. The aspects of a structural driven rational design approach and its implications for further engineering of monomeric TIM variants are discussed. Process development based on a new technology, EnBase®, addresses the relative instability of new variants, compared to its ancestors, for further studies. EnBase® is then applied for the production of 15N isotope labeling of a monomeric TIM variant, A-TIM. Systematical function- and engineering studies on dimeric TIM and monomeric TIM in regards to the hinges of the catalytic loop-6 were conducted to investigate enzyme activity and stability. Both the A178L and P168A were proposed to induce loop-6 closure, a wanted feature for A-TIM variants. The P168A mutants are hardly active, but gave great insight into the catalytic machinery, while the A178L mutants did induce partial loop-6 closure, however in addition, monomeric A178L was destabilized. Homology driven genome mining and subsequent isolation- high throughput (HTP) overexpression of a thermostable UP from the Archaea Aeopyrum pernix was carried out as an example for the production of recombinant proteins. In addition an alternative kinetic method to study the kinetics of UP by means of NMR directly from cell lysate is discussed. The combination of expression libraries and EnBase® in a HTP manner may relieve up the gene-to-product bottleneck. The structural aspects of A. pernix UP are explored by means of simple bioinformatic tools in the last section of this thesis. A thermostable, truncated version of UP was created and its use for protein engineering in the future is explored. The long N-terminal and C-terminal ends of A. pernix UP seem to be involved in stabilizing the dimeric and hexameric structures of UP. However, deletion of the N-terminal end of A. pernix UP yielded a thermostable protein. Overall, the finding in regards to process optimization and HTP expression and optimization and the underlying methods used in the TIM studies and the UP studies are interchangeable.

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La déamidation est la modification post-traductionnelle de l'asparagine (Asn) et de la glutamine (Glu). Elle est communèment observée dans les peptides et les protéines. Il a été démontré que la déamidation limite la durée de vie de ces macromolécules. Dans ce travail, la déamidation de l'asparagine dans des petits peptides et dans l'enzyme triosephosphate isomérase a été modélisée. La déamidation dans la triosephosphate isomérase de mammifères a été observée sur deux sites distincts: Asn15 et Asn71. Asn71 a une vitesse de déamidation plus élevée que Asn15 et moins grande que pour un petit peptide. Il a été suggéré que la déamidation de Asn15 se produit sous l'influence de la déamidation de Asn71. Pour expliquer ces résultats expérimentaux, des simulations de dynamiques moléculaires classiques à l'échelle de la microseconde et des calculs d'énergie libre, de type umbrella sampling, à l'aide de méthodes combinées mécanique quantique/mécanique moléculaire ont été réalisés. Nous montrons que la déamidation séquentielle dans la triosephosphate isomérase est due à la fois à des effets locaux et globaux. Ces résultats apporte une nouvelle perspective sur l'impact de l'ordre structurel sur la vitesse de déamidation Nous avons également déterminé la voie la plus plausible de cette reaction ainsi que l'influence de la variation du pKa, dans la chaîne principale, de la partie amide du résidu adjacent de l'asparagine sur la vitesse de déamidation. En regard de l'importance des variations de pKa dans l'environnement protéique, nous avons élaboré un protocole informatique permettant d'évaluer de manière rapide et précise des pKa . Ce protocole a été appliqué à des petites molécules organiques et nous avons montré qu'il était également applicable à des études relatives à la prédiction de pKa dans les protéines / Deamidation is the posttranslational modification of asparagine (Asn) and glutamine (Glu) residues, which is observed in several proteins and peptides. It has been shown that deamidation limits the lifetime of these macromolecules. In this work, deamidation of asparagine in small peptides and in the enzyme triosephosphate isomerase has been modeled. Deamidation in mammalian triosephosphate isomerase has been observed at two distinct deamidation sites: Asn15 and Asn71. Asn71 deamidates faster than Asn15 and slower than a small peptide. It has been suggested that, deamidation at Asn15 occurs with the influence of deamidated Asn71. In order to explain these experimental findings, microsecond long classical molecular dynamics simulations and free energy calculations using quantum mechanics/molecular mechanics tools combined with umbrella sampling technique have been performed. The sequential deamidation in triosephosphate isomerase has been shown to be related with both global and local effects. These results bring a new perspective to the impact of the high-order structure on deamidation rate. The most plausible route of this reaction was also determined. The pKa shift of backbone amide of the residue adjacent to asparagine has been found to be one of the most crucial factor determining the rate of deamidation. Considering the importance of pKa shifts in protein environment, a computational protocol was suggested in order to obtain accurate and fast pKa predictions. This protocol was applied to small organic molecules, and it has been shown to be applicable to studies concerning aminoacid pKa predictions

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Ce projet a pour but d’évaluer la capacité de la voie des pentoses phosphates (VPP) dans les racines transgéniques de pomme de terre (Solanum tuberosum) modifiées pour exprimer différents niveaux de l'hexokinase (HK) et de la triosephosphate isomérase cytosolique (cTPI). Dans les racines, la VPP alimente la voie de l’assimilation de l’azote en equivalents réducteurs et permet donc la biosynthèse des acides aminés. Le glucose-6-phosphate produit par l’HK est consommé par la partie oxydative de la VPP catalysée par la glucose-6-phosphate déshydrogénase (G6PDH) et la 6-phosphogluconate déshydrogénase (6PGDH). Les changements dans l'expression de HK et cTPI peuvent affecter le fonctionnement de la VPP et les mécanismes qui sont liés à l’utilisation des équivalents réducteurs produits par la VPP, comme l'assimilation de l’azote et la synthèse des acides aminés. Afin d’évaluer l’effet des manipulations génétiques de l’HK et de la cTPI sur l’assimilation de l’azote, nous avons cultivé les racines transgéniques sur des milieux contenant des concentrations élevées (7 mM) ou basses (0,7 mM) de nitrate d’ammonium comme source d’azote. Les résultats montrent que la culture sur un milieu riche en azote induit les activités G6PDH et 6PGDH. Les données montrent que la capacité de la VPP est plus grande avec des niveaux élevés en HK ou en cTPI. Nous avons aussi pu démontrer une plus grande activité spécifique de l’HK dans les conditions pauvres en azote. Ces données ont été complémentées par des mesures des pools d’acides aminés dans les racines transgéniques cultivées sur différents niveaux d’azote. Aucune tendance notable des pools d’acides aminés n’a été remarquée dans les racines modifiées pour leur contenu en HK suggèrant que la manipulation de HK n’affecte pas l'assimilation de l’azote. Dans les racines transgéniques modifiées pour la cTPI, les ratios Gln/Glu et Asn/Asp sont plus élevés chez les clones antisens, indiquant une assimilation de l’azote plus élevée. Ces résultats ont démontré l'activation de l'assimilation de l’azote chez les clones antisens cTPI dans les conditions élevées et basses d’azote alors que la manipulation de l’HK n’affecte pas l’assimilation de l’azote. / This study investigates the capacity of the oxidative pentose phosphate pathway (oxPPP) and nitrogen metabolism in transgenic potato (Solanum tuberosum) roots modified to express different levels of hexokinase (HK) or cytosolic triosephosphate isomerase (cTPI) growing under different nitrogen regimes. The flux of carbon through the oxPPP in cTPI antisense roots is higher than control roots growing under high supply of N. On the other hand, the conversion of Glucose (Glc) to Glucose-6-phosphate (G6P) is higher in roots overexpressing HK than in antisense HK roots growing at a high level of N. Therefore, overexpression of HK or down regulation of cTPI activities in transgenic roots might be compensated by increased C catabolism through the oxPPP. In order to see the affect of HK and cTPI manipulation on N assimilation, the transgenic roots were grown on media with low or high concentration of ammonium nitrate as the N source. The specific activity of the oxPPP enzymes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) were both increased by an increased N supply in HK and cTPI transgenic roots. This is consistent with the provision of reducing equivalents for N assimilation. The data also show that the capacity of the oxPPP is higher in roots with high HK or cTPI activity. We were able to detect higher HK specific activity in N deficient conditions. These data were complemented with measurements of amino acid pools in transgenic roots. No trend in amino acid pools was found in roots modified for HK activity. However, down regulation of cTPI led to higher Gln, Gln/Glu and Asn/Asp ratios, indicating higher assimilation of N. These results demonstrated the activation of N assimilation in cTPI antisense clones while the manipulation of HK is unlikely to affect the N assimilation.

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