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J. Biol. Chem., Vol. 283, Issue 21, 14762-14771, May 23, 2008

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Mutagenesis Studies of Substrate Recognition and Catalysis in the Sortase A Transpeptidase from Staphylococcus aureus^*

Matthew L. Bentley, Erin C. Lamb, and Dewey G. McCafferty¹

From the Department of Biochemistry and Biophysics and the Johnson Research Foundation, The 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) is responsible for anchoring a range of virulence- and colonization-associated proteins to the cell wall. SrtA recognizes substrates that contain a C-terminal LPXTG motif. This sequence is cleaved following the threonine, and an amide bond is formed between the threonine and the pentaglycine cross-bridge of branched lipid II. Previous studies have implicated the β6/β7 loop region of SrtA in LPXTG recognition but have not systematically characterized this domain. To better understand the individual roles of the residues within this loop, we performed alanine-scanning mutagenesis. Val-168 and Leu-169 were found to be important for substrate recognition, and Glu-171 was also found to be important, consistent with its hypothesized role as a Ca²⁺-binding residue. Gly-167 and Asp-170 were dispensable for catalysis, as was Gln-172. The role of Arg-197 in SrtA has been the subject of much debate. To explore its role in catalysis, we used native chemical ligation to generate semi-synthetic SrtA in which we replaced Arg-197 with citrulline, a non-ionizable analog. This change resulted in a decrease of <3-fold in k_cat/K_m, indicating that Arg-197 utilizes a hydrogen bond, rather than an electrostatic interaction. Our results are consistent with a model for LPXTG recognition wherein the Leu-Pro sequence is recognized primarily by hydrophobic contacts with SrtA Val-168 and Leu-169, as well as a hydrogen bond from Arg-197. This model contradicts the previously proposed mechanism of binding predicted by the x-ray crystal structure of SrtA.

Received for publication, February 6, 2008 , and in revised form, March 24, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant AI46611. 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.

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.mccafferty{at}duke.edu.

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				P. R. Race, M. L. Bentley, J. A. Melvin, A. Crow, R. K. Hughes, W. D. Smith, R. B. Sessions, M. A. Kehoe, D. G. McCafferty, and M. J. Banfield Crystal Structure of Streptococcus pyogenes Sortase A: IMPLICATIONS FOR SORTASE MECHANISM J. Biol. Chem., March 13, 2009; 284(11): 6924 - 6933. [Abstract] [Full Text] [PDF]