Computational thermostabilization of an enzyme.
Thermostabilizing an enzyme while maintaining its activity for industrial or biomedical applications can be difficult with traditional selection methods. We describe a rapid computational approach that identified three mutations within a model enzyme that produced a 10 degrees C increase in apparent melting temperature T(m) and a 30-fold increase in half-life at 50 degrees C, with no reduction in catalytic efficiency. The effects of the mutations were synergistic, giving an increase in excess of the sum of their individual effects. The redesigned enzyme induced an increased, temperature-dependent bacterial growth rate under conditions that required its activity, thereby coupling molecular and metabolic engineering.
Korkegian
Aaron
A
Division of Basic Sciences, Fred Hutchinson Cancer Research Center (FHCRC), 1100 Fairview Avenue North, Seattle, WA 98109, USA.
Black
Margaret E
ME
Baker
David
D
Stoddard
Barry L
BL
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D043525:Cytosine Deaminase / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009010:Monte Carlo Method; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012984:Software; D013696:Temperature; D013816:Thermodynamics; D014170:Transformation, Genetic; D015003:Yeasts / Q000201:enzymology
D043525:Cytosine Deaminase