Design of Ca2+-independent Staphylococcus aureus sortase A mutants.
The catalytic activity of Staphylococcus aureus sortase A (SaSrtA) is dependent on Ca(2+), because binding of Ca(2+) to Glu residues distal to the active site stabilizes the substrate binding site. To obtain Ca(2+)-independent SaSrtA, we substituted two Glu residues in the Ca(2+)-binding pocket (Glu(105) and Glu(108)). Although single mutations decreased SaSrtA activity, mutations of both Glu(105) and Glu(108) resulted in Ca(2+)-independent activity. Kinetic analysis suggested that the double mutations affect the substrate binding site, without affecting substrate specificity. This approach will allow us to develop SaSrtA variants suitable for various applications, including in vivo site-specific protein modification and labeling.
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. hirakawa@bio.t.u-tokyo.ac.jp
Hidehiko
H
Hirakawa
Suguru
S
Ishikawa
Teruyuki
T
Nagamune
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018698:Glutamic Acid; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D018698:Glutamic Acid; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D002118:Calcium