title: Development of small designer aldolase enzymes: catalytic activity, folding, and substrate specificity.
abstract: Small (24-35 amino acid residues) peptides that catalyze carbon-carbon bond transformations including aldol, retro-aldol, and Michael reactions in aqueous buffer via an enamine mechanism have been developed. Peptide phage libraries were created by appending six randomized amino acid residues to the C-terminus or to the N-terminus of an 18-mer alpha-helix peptide containing lysine residues. Reaction-based selection with 1,3-diketones was performed to trap the amino groups of reactive lysine residues that were necessary for the catalysis via an enamine mechanism by formation of stable enaminones. The selected 24-mer peptides catalyzed the reactions with improved activities. The improved activities were correlated with improved folded states of the peptides. The catalyst was then improved with respect to substrate specificity by appending a phage display-derived substrate-binding module. The resulting 35-mer peptide functioned with a significant proportion of the catalytic proficiency of larger protein catalysts. These results indicate that small designer enzymes with good rate acceleration and excellent substrate specificity can be created by combination of design and reaction-based selection from libraries.
authors.affiliation: The Skaggs Institute for Chemical Biology and Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. ftanaka@scripps.edu
authors.forename: Fujie
authors.initials: F
authors.lastname: Tanaka
authors.forename: Roberta
authors.initials: R
authors.lastname: Fuller
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Barbas
mesh_terms: D000447:Aldehydes / Q000737:chemistry; D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D002384:Catalysis; D015195:Drug Design; D004789:Enzyme Activation; D005634:Fructose-Bisphosphate Aldolase / Q000138:chemical synthesis* / Q000737:chemistry; D007659:Ketones / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D013379:Substrate Specificity; D013780:Tetroses / Q000737:chemistry; D047090:beta-Lactams / Q000737:chemistry
chemical_list: D000447:Aldehydes; D000588:Amines; D007659:Ketones; D019151:Peptide Library; D010455:Peptides; D013780:Tetroses; D047090:beta-Lactams; C116609:3-hydroxybutanal; D005634:Fructose-Bisphosphate Aldolase; C073321:erythrose