Design of Ca2+-independent Staphylococcus aureus sortase A mutants

Authors

  • Hidehiko Hirakawa,

    Corresponding author
    1. Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; telephone: +81-3-5841-7356; fax: +81-3-5841-8657
    • Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; telephone: +81-3-5841-7356; fax: +81-3-5841-8657
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  • Suguru Ishikawa,

    1. Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; telephone: +81-3-5841-7356; fax: +81-3-5841-8657
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  • Teruyuki Nagamune

    1. Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; telephone: +81-3-5841-7356; fax: +81-3-5841-8657
    2. Department of Bioengineering, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
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  • Conflict of interest: Nothing to declare.

Abstract

The catalytic activity of Staphylococcus aureus sortase A (SaSrtA) is dependent on Ca2+, because binding of Ca2+ to Glu residues distal to the active site stabilizes the substrate binding site. To obtain Ca2+-independent SaSrtA, we substituted two Glu residues in the Ca2+-binding pocket (Glu105 and Glu108). Although single mutations decreased SaSrtA activity, mutations of both Glu105 and Glu108 resulted in Ca2+-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. Biotechnol. Bioeng. 2012; 109: 2955–2961. © 2012 Wiley Periodicals, Inc.

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