Abstract
Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with a LPXTG motif. Sortase cleaves polypeptides between the threonine and the glycine of the LPXTG motif. The carboxyl group of threonine is subsequently amide-linked to the amino group of peptidoglycan cross-bridges. The three-dimensional structure of sortase revealed the close proximity of the catalytic site residue cysteine 184 with histidine 120; however, no structural evidence for a thiolate-imidazolium ion pair could be detected. We report that alanine substitution of either cysteine 184 or histidine 120 abolishes in vivo and in vitro sorting reactions. Further, alanine substitution of tryptophan 194, a residue that is in close proximity of histidine 120, reduces the transpeptidase activity of sortase. These results suggest a model whereby sortase forms a thiolate-imidazolium ion pair for the catalysis of its transpeptidation reaction and that the position of tryptophan 194 assists in the formation of this ion pair.
Highlights
Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with a LPXTG motif
Together these results suggest that the single cysteine residue of sortase performs the nucleophilic attack at the peptide bond between the threonine and the glycine of the LPXTG motif
Effect of Alanine Substitutions of Sortase on in Vivo Surface Protein Anchoring in S. aureus—To examine the role of the conserved residues of sortase in surface protein anchoring to the cell wall of S. aureus, site-directed mutagenesis was performed to substitute each codon, i.e. leucine 97, histidine 120, tyrosine 153, cysteine 184, and tryptophan 194, with an alanine codon
Summary
CYSTEINE 184 AND HISTIDINE 120 OF SORTASE FORM A THIOLATE-IMIDAZOLIUM ION PAIR FOR CATALYSIS*. Alanine substitution of tryptophan 194, a residue that is in close proximity of histidine 120, reduces the transpeptidase activity of sortase These results suggest a model whereby sortase forms a thiolate-imidazolium ion pair for the catalysis of its transpeptidation reaction and that the position of tryptophan 194 assists in the formation of this ion pair. Alanine substitution of tryptophan 194, a residue that is in close proximity of histidine 120, reduces the transpeptidase activity of sortase, whereas alanine substitution at Leu causes little or no effect
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