Tübingen, Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Freiburg (Breisgau), Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Freiburg (Breisgau), Universiẗat, Diss., 2003.

No description available.

Characterization of allosteric properties of phosphofructokinase from the extreme thermophile Thermus thermophilus (TtPFK) using thermodynamic linkage analysis revealed several peculiarities. Inhibition and activation of Fru-6-P binding by the allosteric effectors phosphoenolpyruvate (PEP) and MgADP are entropically-driven in TtPFK. It is also curious that PEP binding affinity is unusually strong in TtPFK when compared to PFKs from Escherichia coli, Bacillus stearothermophilus, and Lactobacillus delbrueckii, while the magnitude of the allosteric inhibition by PEP is much smaller in TtPFK. In an effort to understand the source of weak inhibition, a putative network of residues between the allosteric site and the nearest active site was identified from the three-dimensional structures of BsPFK. Three of the residues in this network, D59, T158, and H215, are not conserved in TtPFK, and, due to their nature (N59, A158, S215), are unlikely to be involved in the same non-covalent interactions seen in BsPFK. The triple chimeric substitution N59D/A158T/S215H, results in a 2.5 kcal mol-1 increase in the coupling free energy, suggesting that the region containing these residues may be important for propagation of inhibitory response. The individual substitutions at each position resulted in an increase in the coupling free energy, and the double substitutions displayed additivity of these changes. The chimeric substitution made at N59 suggests that the polar nature of the asparagine at position 59 is key for the enhanced binding of PEP. The non-conserved R55 was found to be particularly important for the enhanced binding of PEP in TtPFK, as chimeric substitutions R55G and R55E resulted in a 3.5 kcal mol-1 and 4.5 kcal mol-1 decrease in the binding affinity for PEP, respectively. Our results also confirm the observations previously made in PFKs from E. coli and B. stearothermophilus, that the ability of the effector to bind is independent of its ability to produce allosteric response. We show that several substitutions result in a decrease in binding affinity of PEP to TtPFK, while dramatically enhancing its ability to inhibit (N59D, R55G, R55E). Similarly, some substitutions, like S215H and A158T show an enhanced inhibition by PEP, while having no effect on its binding affinity.

Greifswald, Universiẗat, Diss., 2002.

The class Cyanidiophyceae (the "cyanidia") includes three genera, the walled Cyanidium and Galdieria and the "naked" Cyandioschyzon. All of these algae are unicellular and asexual and live at high temperature and low pH. The cyanidia grow optimally at a pH of 2-3 but can tolerate a higher pH and lower their surrounding pH if it is above the optimal level. They can also tolerate high concentrations of potential toxins that are often found in their natural environments. This thesis shows that strains of cyanidia from Yellowstone National Park and other geographic locations have differing abilities to lower their surrounding pH and tolerate environmental toxins that are found in many environments in which they live. These unique characteristics of this class of algae allow them to be optimally adapted for life in extreme environments with few competitors. This thesis includes unpublished co-authored material.

High catalytic activity and a broad substrate range are characteristic of P450 fusion enzymes of the CYP102A class. P450 BM3 (CYP102A1, BM3) is a paradigm for the P450 fusion enzymes and is accredited with the highest monooxygenase activity in the P450 superfamily, a property which has led to its engineering and exploitation for biotechnologically valuable oxidation reactions. Initial research in the thesis focused on characterisation of a novel P450-redox partner fusion enzyme from the thermophilic fungus Myceliophthora thermophila (CYP505A30, P450MT1). Sequence alignments revealed a P450 domain and a diflavin P450 reductase domain with high sequence similarity to BM3’s domains (41% and 31% amino acid identity, respectively). The purified 118 kDa protein is soluble and exhibits characteristic P450 spectral properties, giving a Soret absorption shift to 450 nm upon binding CO to its ferrous heme iron. Binding titrations of intact P450 MT1 and its expressed P450 (heme) domain with fatty acid substrates and imidazole-based inhibitors revealed type I (blue) and II (red) Soret shifts, respectively, typical of other members of the P450 superfamily, and enabled determination of substrate binding constants. HPLC analysis confirmed stoichiometric amounts of bound FAD and FMN cofactors. Subsequent kinetic and biochemical studies included stopped-flow kinetic experiments showing that NADPH-dependent reduction of P450 MT1’s FAD cofactor occurs with a rate constant of ~150 s-1 at 20 °C. P450 MT1 has an unconventional substrate hydroxylation profile for saturated fatty acids. It hydroxylates these substrates predominantly at positions ω-1, ω-2 and ω-3. However, an unusual property of this enzyme is observed in its strong preference (~85% of total converted product) for either the ω-1 or the ω-2 position on odd and even chain length fatty acids, respectively. However, it displays higher selectivity for branched chain fatty acids over straight chain fatty acids, e.g. for the substrate iso-myristic acid, similar to BM3’s properties. Other work done focused on biophysical characterisation of the model P450-reductase fusion enzyme P450 BM3 from Bacillus megaterium. A combination of alternative structural techniques to X-ray crystallography were used to characterise the enzyme. More specifically, electron microscopy (EM) and nuclear magnetic resonance (NMR) were used to gain greater insights into the intimate associations of the enzyme monomers in BM3’s dimeric structure. These studies led to the first structural insights into how P450 BM3’s dimeric complex is organised. Dimerisation in BM3 arises predominantly from self-association of the enzyme’s FAD domains, and wild-type and mutant BM3 FAD domain forms were also characterised. Key FAD domain mutations that prevented intra-/inter-monomer disulphide bond formation facilitated the crystallization and determination of the FAD domain structure, the final part of the BM3 enzyme to have its three dimensional structure resolved. Data reported in this thesis give new insights into the biochemistry of biotechnologically important P450 monooxygenase enzymes from mesophilic and thermophilic microorganisms.

Zugl.: Aachen, Techn. Hochsch., Diss., 2009

Universiẗat, Diss., 2003--Tübingen.