title: Ca2+ -independent sortase-A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli.
abstract: A Staphylococcus aureus transpeptidase, sortase A (SrtA), which catalyzes a peptide ligation with high substrate specificity, is a useful tool to site-specifically attach proteinaceous/peptidic functional molecules to target proteins. However, its strong Ca(2+) dependency makes SrtA difficult for use under low Ca(2+) concentrations and in the presence of Ca(2+)-binding substances. To overcome this problem, we designed a SrtA mutant that Ca(2+)-independently demonstrates a high catalytic activity. The heptamutant (P94R/E105K/E108A/D160N/D165A/K190E/K196T), which resulted from a combination of known mutations at the Ca(2+) -binding site and around the substrate-binding site, successfully catalyzed a selective protein-protein ligation in the cytoplasm of Escherichia coli. Selective protein modification in living cells is a promising approach for investigating cellular events and regulating cell functions. This SrtA mutant may prove to be a versatile tool for adding new functionalities to proteins of interest by incorporating functional proteins and chemically modified peptides in living cells, which usually retain low Ca(2+) concentrations.
authors: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan. hirakawa@bio.t.u-tokyo.ac.jp.
authors: Hidehiko
authors: H
authors: Hirakawa
authors: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.
authors: Suguru
authors: S
authors: Ishikawa
authors: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.|Department of Bioengineering, School of Engineering, The University of Tokyo, Tokyo, Japan.
authors: Teruyuki
authors: T
authors: Nagamune
mesh_terms: D019881:Aminoacyltransferases / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002118:Calcium; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011506:Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D002118:Calcium
keywords: Calcium ion; Mutant; Protein ligation; Sortase A; Staphylococcus aureus