RESUMO
Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (ΔTm ≥ +3 °C). By combining mutations, a 10-fold mutant was generated with a Tm of 94 °C (+51 °C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a Tm of 88 °C (+45 °C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.
Assuntos
Álcool Desidrogenase/química , Escherichia coli/genética , Mutação , Engenharia de Proteínas/métodos , Temperatura de Transição , Álcool Desidrogenase/genética , Computadores Moleculares , Cristalização , Estabilidade Enzimática , Biblioteca Gênica , Cinética , Conformação Proteica , Saccharomycetales/enzimologiaRESUMO
Monooxygenases (MOs) face the challenging reaction of an organic target, oxygen and a cofactor - most commonly heme or flavin. To correctly choreograph the substrates spatially and temporally, MOs evolved a variety of strategies, which involve structural flexibility. Besides classical domain and loop movements, flavin-containing MOs feature conformational changes of their flavin prosthetic group and their nicotinamide cofactor. With similar mechanisms emerging in various subclasses, their generality and involvement in selectivity are intriguing questions. Cytochrome P450 MOs are often inherently plastic and large movements of individual segments throughout the entire structure occur. As these complicated and often unpredictable movements are largely responsible for substrate uptake, engineering strategies for these enzymes were mostly successful when randomly mutating residues across the entire structure.