Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase.
Variation in gene sequences generated by directed evolution approaches often does not assure a minimalist design for obtaining a desired property in proteins. While screening for enhanced thermostability, structural information was utilized in selecting mutations that are generated by error-prone PCR. By this approach we have increased the half-life of denaturation by 300-fold compared to the wild-type Bacillus subtilis lipase through three point mutations generated by only two cycles of error-prone PCR. At lower temperatures the activity parameters of the thermostable mutants are unaltered. High-resolution crystal structures of the mutants show subtle changes, which include stacking of tyrosine residues, peptide plane flipping and a better anchoring of the terminus, that challenge rational design and explain the structural basis for enhanced thermostability. The approach may offer an efficient and minimalist solution for the enhancement of a desired property of a protein.
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Priyamvada
P
Acharya
Eerappa
E
Rajakumara
Rajan
R
Sankaranarayanan
Nalam M
NM
Rao
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D019143:Evolution, Molecular; D006358:Hot Temperature; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D017354:Point Mutation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D008049:Lipase