Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcusfuriosus Phosphoglucose Isomerase
| Autor: | Kalyanasundaram Subramanian, Svetlana E. Sedelnikova, Servé W. M. Kengen, John van der Oost, Karolina Mitusińska, Henk-Jan Joosten, Peter J. Schaap, Vitor A. P. Martins dos Santos, Sjon Hendriks, John Raedts, Feras M. Almourfi, Patrick J. Baker, Wilfred R. Hagen, Artur Góra |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Glucose-6-phosphate isomerase Protein Conformation Mutant lcsh:QR1-502 cupin phosphoglucose isomerase Molecular Dynamics Simulation 010402 general chemistry medicine.disease_cause Protein Engineering 01 natural sciences Biochemistry Microbiology Cofactor lcsh:Microbiology Article solvent access 03 medical and health sciences Microbiologie medicine Systems and Synthetic Biology Pyrococcus furiosus Enzyme Inhibitors Molecular Biology VLAG chemistry.chemical_classification Mutation Systeem en Synthetische Biologie Manganese biology Chemistry Glucose-6-Phosphate Isomerase Temperature Water BacGen Comulator Protein engineering biology.organism_classification 0104 chemical sciences Amino acid 030104 developmental biology Enzyme biology.protein Mutant Proteins |
| Zdroj: | Biomolecules 9 (2019) 6 Biomolecules Biomolecules, Vol 9, Iss 6, p 212 (2019) Volume 9 Issue 6 Biomolecules, 9(6) |
| ISSN: | 2218-273X |
| Popis: | The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations. |
| Databáze: | OpenAIRE |
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