Abstract
Surface proteins of Staphylococcus aureus are anchored to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Sortase A cleaves surface proteins between the threonine (T) and the glycine (G) residues of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine at the C-terminal end of polypeptides and the amino group of pentaglycine cross-bridges of cell wall peptidoglycan. Previous work showed that Cys(184) and His(120) of sortase A are absolutely essential for catalysis; however an active site thiolateimidazolium ion pair may not be formed. The three-dimensional crystal structure of sortase A revealed that Arg(197) is located in close proximity to both the active site Cys(184) and the scissile peptide bond between threonine and glycine. We show here that substitution of Arg(197) with alanine, lysine, or histidine severely reduced sortase A function both in vivo and in vitro, whereas Asn(98), which had earlier been implicated in hydrogen bonding to His(120), was found to be dispensable for catalysis. As the structural proximity of Arg(197) and Cys(184) is conserved in sortase enzymes and as ionization of the Cys(184) sulfhydryl group seems required for sortase activity, we propose that Arg(197) may function as a base, facilitating thiolate formation during sortase-mediated cleavage and transpeptidation reactions.
Highlights
Surface proteins of Staphylococcus aureus are anchored to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif
As the structural proximity of Arg197 and Cys184 is conserved in sortase enzymes and as ionization of the Cys184 sulfhydryl group seems required for sortase activity, we propose that Arg197 may function as a base, facilitating thiolate formation during sortase-mediated cleavage and transpeptidation reactions
As the structural proximity of Arg197 and Cys184 is conserved in sortase enzymes [32, 33] and as ionization of the Cys184 sulfhydryl group appears to be required for sortase activity [12, 26], we propose that Arg197 may act as a base, facilitating thiolate formation during sortase A cleavage and transpeptidation reactions
Summary
Surface proteins of Staphylococcus aureus are anchored to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Sortase A cleaves surface proteins between the threonine (T) and the glycine (G) residues of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine at the C-terminal end of polypeptides and the amino group of pentaglycine crossbridges of cell wall peptidoglycan. The threedimensional crystal structure of sortase A revealed that Arg197 is located in close proximity to both the active site Cys184 and the scissile peptide bond between threonine and glycine. Gram-positive bacteria use cell wall-anchored surface proteins to invade, colonize, and replicate within their hosts [3,4,5], as polypeptides can promote bacterial attachment to specific host molecules or prevent escape from host immune responses [6, 7]. Tel.: 773-834-9060; Fax: 773-834-8150; E-mail: oschnee@ delphi.bsd.uchicago.edu
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