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J. Biol. Chem., Vol. 282, Issue 9, 6571-6581, March 2, 2007

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Engineering the Substrate Specificity of Staphylococcus aureus Sortase A

THE 6/7 LOOP FROM SrtB CONFERS NPQTN RECOGNITION TO SrtA^*

Matthew L. Bentley, Helena Gaweska, Joseph M. Kielec, and Dewey G. McCafferty¹

From the Department of Biochemistry and Biophysics and the Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and the Department of Chemistry, Duke University, Durham, North Carolina 27708

The Staphylococcus aureus transpeptidase Sortase A (SrtA) anchors virulence and colonization-associated surface proteins to the cell wall. SrtA selectively recognizes a C-terminal LPXTG motif, whereas the related transpeptidase Sortase B (SrtB) recognizes a C-terminal NPQTN motif. In both enzymes, cleavage occurs after the conserved threonine, followed by amide bond formation between threonine and the pentaglycine cross-bridge of cell wall peptidoglycan. Genetic and biochemical studies strongly suggest that SrtA and SrtB exhibit exquisite specificity for their recognition motifs. To better understand the origins of substrate specificity within these two isoforms, we used sequence and structural analysis to predict residues and domains likely to be involved in conferring substrate specificity. Mutational analyses and domain swapping experiments were conducted to test their function in substrate recognition and specificity. Marked changes in the specificity profile of SrtA were obtained by replacing the 6/7 loop in SrtA with the corresponding domain from SrtB. The chimeric 6/7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTN-containing substrates, with a of 0.0062 ± 0.003 M^-1 s^-1. This enzyme was unable to perform the transpeptidation stage of the reaction, suggesting that additional domains are required for transpeptidation to occur. The overall catalytic specificity profile () of SrtLS was altered 700,000-fold from SrtA. These results indicate that the 6/7 loop is an important site for substrate recognition in sortases.

Received for publication, November 13, 2006 , and in revised form, December 21, 2006.

^* This work was supported by National Institutes of Health Grant AI46611 (to D. G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Chemistry, Duke University, B120 LSRC Bldg., Box 90317, Durham, NC 27708-0317. Tel.: 919-660-1516; Fax: 919-668-5483; E-mail: dewey{at}duke.edu.

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