Directed evolution of a histone acetyltransferase--enhancing thermostability, whilst maintaining catalytic activity and substrate specificity.
Histone acetylation plays an integral role in the epigenetic regulation of gene expression. Transcriptional activity reflects the recruitment of opposing classes of enzymes to promoter elements; histone acetyltransferases (EC 2.3.1.48) that deposit acetyl marks at a subset of histone residues and histone deacetylases that remove them. Many histone acetyltransferases are difficult to study in solution because of their limited stability once purified. We have developed a directed evolution protocol that allows the screening of hundreds of histone acetyltransferase mutants for histone acetylating activity, and used this to enhance the thermostability of the human P/CAF histone acetyltransferase. Two rounds of directed evolution significantly stabilized the enzyme without lowering the catalytic efficiency and substrate specificity of the enzyme. Twenty-four variants with higher thermostability were identified. Detailed analysis revealed twelve single amino acid mutants that were found to possess a higher thermostability. The residues affected are scattered over the entire protein structure, and are different from mutations predicted by sequence alignment approaches, suggesting that sequence comparison and directed evolution methods are complementary strategies in engineering increased protein thermostability. The stabilizing mutations are predominately located at surface of the enzyme, suggesting that the protein's surface is important for stability. The directed evolution approach described in the present study is easily adapted to other histone modifying enzymes, requiring only appropriate peptide substrates and antibodies, which are available from commercial suppliers.
Leemhuis
Hans
H
Department of Biochemistry, University of Cambridge, UK.
Nightingale
Karl P
KP
Hollfelder
Florian
F
D002384:Catalysis; D019020:Directed Molecular Evolution; D004795:Enzyme Stability / Q000235:genetics*; D051548:Histone Acetyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D050505:Mutant Proteins; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature
D006657:Histones; D050505:Mutant Proteins; D051548:Histone Acetyltransferases