Schwab, Thomas and Sterner, Reinhard (2011) Stabilization of a Metabolic Enzyme by Library Selection in Thermus thermophilus. CHEMBIOCHEM, 12 (10). pp. 1581-1588. ISSN 1439-4227,
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The anthranilate phosphoribosyl transferase from the hyper-thermophilic archaeon Sulfolobus solfataricus (sAnPRT, encoded by strpD), which catalyzes the third step in tryptophan biosynthesis, is a thermostable homodimer with low enzymatic activity at room temperature. We have combined two mutations leading to the monomerization and two mutations leading to the activation of sAnPRT. The resulting "activated monomer" sAnPRT-I36E-M47D + D83G-F149S, which is much more labile than wild-type sAnPRT, was stabilized by a combination of random mutagenesis and metabolic library selection using the extremely thermophilic bacterium Thermus thermophilus as host. This approach led to the identification of five mutations that individually increased the thermal stability of sAnPRT-I36E-M47D + D83G-F149S by 1 to 8 degrees C, and by 13 degrees C when combined. The beneficial exchanges were located in different parts of the protein structure, but none of them led to the "re-dimerization" of the enzyme. We observed a negative correlation between thermal stability and catalytic activity of the mutants; this suggests that conformational flexibility is required for catalysis by sAnPRT.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | ADAPTIVE PROTEIN EVOLUTION; ANTHRANILATE PHOSPHORIBOSYLTRANSFERASE; SULFOLOBUS-SOLFATARICUS; EXTREME THERMOPHILE; DIRECTED EVOLUTION; 3-ISOPROPYLMALATE DEHYDROGENASE; CRYSTAL-STRUCTURE; STABILITY; MUTAGENESIS; ADAPTATION; biosynthesis; enzymes; phosphoribosyl-transferase; protein stability; Sulfolobus solfataricus |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Reinhard Sterner |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 05 Jun 2020 08:52 |
| Last Modified: | 05 Jun 2020 08:52 |
| URI: | https://pred.uni-regensburg.de/id/eprint/20555 |
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