Computer-aided design of the stability of pyruvate formate-lyase from Escherichia coli by site-directed mutagenesis.
Using computer-aided design of single-site mutations, three amino acid residues determined by changes in folding free energy between wild-type (wt) and mutant proteins were exchanged to enhance the stability of pyruvate formate-lyase (PFL). The mutant enzymes were tested for properties such as optimum temperature, optimum pH, kinetic parameters, and stability to temperature. There were two mutant variants, Glu336Cys and Glu400Ile, that exhibited increased thermostability as compared to the wt enzyme. The melting temperatures (T(m), the temperature at which 50% inactivation occurs after heat treatment for 20 min) of Glu336Cys and Glu400Ile increased by 3.7 and 2.2 respectively. They also showed an increase in half life of about 1.80 and 2.21-fold, whereas Ala273Cys showed a slight decrease as compared with the wt enzyme.
Yang
Deng-Feng
DF
Guangxi Academy of Sciences, Nanning, China.
Wei
Yu-Tuo
YT
Huang
Ri-Bo
RB
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017076:Computer-Aided Design; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D013696:Temperature
D029968:Escherichia coli Proteins; D000123:Acetyltransferases; C022560:formate C-acetyltransferase