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Projekt
How general are the determinants of stability and activity? An evolutionary study with perturbed beta-lactamases
Antragsteller
Professor Dr. Kristian Müller
Fachliche Zuordnung
Biochemie
Förderung
Förderung von 2006 bis 2011
Projektkennung
Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 30531752
Tailoring enzymes for specific needs in industry and medicine is a major goal of protein engineering efforts. Red and white biotechnology applications request optimized enzymes typically not provided by natural resources. In contrast, natural enzymes which inactivate antibiotics pose a major threat to human health and can adapt to new compounds at a fast pace. Both, industrial needs and superbugs demand an understanding of how proteins may evolve to higher stability against different types of stress.Until recently controversy surrounded whether activity at high temperatures is compatible with activity at low temperatures. In sharp contrast to this debate we have demonstrated that directed evolution of activity at physiological temperatures can be used to generate thermostable enzymes. The core of this finding is that general stabilizing mutations exist for enzyme structures and that they act independently of the perturbation. This implies the novel perspective that truncation of a few terminal amino acids is a stress which can be related to increased heat. Consequently, we propose to study this effect in detail starting with TEM ß-lactamase structures which are perturbed by truncation, removal of the disulfide bridge, or the presence of substrate broadening mutations. These enzyme variants will be evolved for activity by metabolic selection. DNA recombination will be achieved with our predictable and reproducible nucleotide exchange and excision technology (NExT) DNA shuffling. Comparing optimized enzymes as well as enzymes bearing only key mutations in biochemical and biophysical studies will answer the question of which type of stabilizing mutations evolve and to what extent the evolutionary compensation of one stress factor helps in the context of another challenge.These studies will deepen our understanding of the evolutionary scope of structure-function relations in order to predict microbial threats and to streamline optimization strategies. In addition, the generated optimized ß-lactamases may serve as versatile reporter enzymes and lead compounds for prodrug activation therapies.
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