The research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study.... Show moreThe research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study. Enzymes inherently embody a delicate balance between activity and stability, and the acquisition of new enzymatic functions is often accompanied by trade-offs, such as decreased stability or reduction of the original activity. Probing evolutionary adaptability of BlaC with laboratory evolution in combination with structural characterization can provide information about the mechanisms of rapid adaptations observed for β-lactamases in the clinic. The role of temperature as a conventional selection pressure in such evolutionary adaptation is unclear. The cooperative nature of enzyme unfolding over a narrow temperature trajectory raises the question whether evolution at temperatures well below the melting point is influenced by temperature. The approach used in this work to answer these questions is by simulating evolution under different selection pressures and characterize the variant enzymes in terms of activity, structure, dynamics and melting temperature. The research makes clear how enzyme kinetics and dynamics vary with different selection pressures and maps the evolutionary path that enzymes may take. The underlying structural mechanisms are established to provide a rationale for the observed effects. Show less
β-Lactamases are enzymes that can break down β-lactam substrates, such as antibiotics, preventing the use of these antibiotics for the treatment of various infectious diseases. However, some... Show moreβ-Lactamases are enzymes that can break down β-lactam substrates, such as antibiotics, preventing the use of these antibiotics for the treatment of various infectious diseases. However, some compounds, β-lactamase inhibitors, can block these enzymes allowing for possible treatments using a combination of antibiotic and inhibitor. BlaC is the β-lactamase of Mycobacterium tuberculosis, the bacteria that cause tuberculosis, and is used as a model for protein evolution. To understand if and how BlaC can develop resistance against certain inhibitors we studied the evolutionary adaptability of this enzyme. We used laboratory evolution and various biochemical techniques to characterize several mutations in BlaC and subsequently tested the effect of combining mutations. One of the findings is that BlaC can easily become less sensitive to the inhibitor sulbactam by partially blocking the entrance to the active site. Interestingly, this was accompanied by increased sensitivity to another inhibitor, avibactam, that could not be compensated for by other mutations.Generally, Escherichia coli bacteria are used to test the effects of BlaC variants in cells, as they are easy and safe to use in the lab. We show that results obtained for E. coli can be extrapolated to conditions that resemble tuberculosis disease in humans: the M. marinum infection model of zebrafish. All these findings are of interest for the future development of combination therapies to treat tuberculosis. Show less
Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity... Show morePhospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity and specificity. The relation between the activity and dynamics of the N-terminal domains of PLAAT3 and PLAAT4 was studied. PLAAT3 has a much higher melting temperature and exhibits less nanosecond and millisecond dynamics in the active site, in particular in loop L2(B6), as shown by NMR spectroscopy and molecular dynamics calculations. Swapping the L2(B6) loops between the two PLAAT enzymes results in strongly increased phospholipase activity in PLAAT3 but no reduction in PLAAT4 activity, indicating that this loop contributes to the low activity of PLAAT3. The results show that, despite structural similarity, protein dynamics differ substantially between the PLAAT variants, which can help to explain the activity and specificity differences. Show less
The work described in this thesis focuses on the application of various NMR techniques to the study of interactions between proteind and small molecules and proteins and peptides, incluiding the... Show moreThe work described in this thesis focuses on the application of various NMR techniques to the study of interactions between proteind and small molecules and proteins and peptides, incluiding the well-established classical NMR approaches, and the precently developed paramagnetic NMR methods. computational tools have been used to complement and visualized the experimental data. Show less
The processes of glycosidic bond formation and destruction are a central theme in glycochemistry and glycobiology, and form the basis of the research described in this Thesis. In the first part,... Show moreThe processes of glycosidic bond formation and destruction are a central theme in glycochemistry and glycobiology, and form the basis of the research described in this Thesis. In the first part, studies towards the stereoselective construction of two complex bacterial oligosaccharide fragments are described. These fragments contain mannuronic acid residues connected through a beta-linkage, which is amongst the most challenging linkages to construct synthetically. In the second part, the use of mannuronic acid building blocks in the automated synthesis of alginates using solid-phase chemistry is presented. Using a second-generation carbohydrate synthesizer, alginate fragments up to 12 residues were assembled with high stereoselectivity. Using the same automated set-up, fragments of hyaluronic acid of up to 15 residues were synthesized. In the third part, 2-deoxy-2-fluoroglucosides are investigated as activity-based probes for glucocerebrosidase, a retaining beta-glucosidase enzyme. Show less
Skov, J.; Gaudin, M.; Podbevsek, P.; Olsthoorn, R.R.C.L.; Petersen, M. 2012
Undoubtedly, grapes and wine are globally the most important fruit and food commodities, respectively. The first objective of this research is to optimize an extraction protocol suitable for grape... Show moreUndoubtedly, grapes and wine are globally the most important fruit and food commodities, respectively. The first objective of this research is to optimize an extraction protocol suitable for grape metabolic profiling followed by the application of that protocol to perform metabolic characterization of grape cultivar, wine types, and vintage using NMR spectroscopy in combination with chemometrics methods. This approach was also used to study different physiological processes in grapevine like ripening of berry and resistance against fungal pathogen. Metabolic characterization of different white wines to highlight the metabolites responsible for sensory attributes was also carried out. Another task of this research was to correlate the metabolic profiling data from grapes and wine with the bioactivity data using various multivariate regression models. Show less
It has been shown by this thesis that plant metabolomics is a promising tool for studying the interaction between B. rapa and pathogenic fungi. It gives a picture of the plant metabolites during... Show moreIt has been shown by this thesis that plant metabolomics is a promising tool for studying the interaction between B. rapa and pathogenic fungi. It gives a picture of the plant metabolites during the interaction. Brassica rapa has many defense related compounds such as glucosinolates, IAA, phenylpropanoids and flavonoids and even some primary metabolites including amino and organic acids which might play role in resistance against pathogens. These metabolic pathways undergo rerouting during the infection and the required precursors for the defense compounds might be formed. The rerouting of fluxes at the level of chorismate seems to play a role in resistance of B. rapa to pathogenic fungi. Resistance involves a very high level of complexity. Understanding resistance thus requires studies of different combinations of cultivars and microorganisms. By taking a systems biology approach and measuring on the level of transcriptome, proteome and metabolome and analyzing the large data sets from interactions of different cultivars with different fungi in combination with data on susceptibility by means of multivariate analysis might give a better insight in the defense response of the plant. As we used different cultivars in our study, the fungal infection and also treatment with signal molecules induced secondary metabolites that branch from the chorismate pathway including phenylpropanoids, flavonoids, IAA and indole glucosinolates. Even with using different cultivars from B. rapa, all cultivars showed enhanced accumulation of secondary metabolites derived from the chorismate pathway. Phenylpropanoids, benzoyl glucosinolates, flavonoids and indole glucosinolates should be the target compounds for genetic engineering research. By using genetic engineering techniques, one can breed new cultivars that are characterized by higher levels of these secondary metabolites. Show less
The biological processes that are the basis of all life forms are mediated largely by protein-protein interactions. The protein complexes involved in these interactions can be categorised by their... Show moreThe biological processes that are the basis of all life forms are mediated largely by protein-protein interactions. The protein complexes involved in these interactions can be categorised by their affinity, which results in a range from static to transient complexes. Electron transfer complexes, which have to combine high turn-over with specificity are typically transient complexes. The transient complexes under investigation in this work are all part of the photosynthetic redox chain, in which electrons are transferred from membrane bound cytochrome f (cytf) to either plastocyanin (Pc) or cytochrome c6 (cytc6). This work describes the investigation of several aspects of transient protein-protein interactions that are applicable to many other transient complexes. The use of NMR as a tool of choice for these studies has proven it an invaluable technique that provides insight in rules that govern the world of transient complex formation. New approaches such as PRE and the creation of ensembles of structures are investigated. They will hopefully lead to a full understanding of dynamics and the variety of ways that transient complexes use dynamics to perform their function in the cell. Show less
