Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus

J Biol Chem. 2008 May 23;283(21):14762-71. doi: 10.1074/jbc.M800974200. Epub 2008 Mar 28.


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 beta6/beta7 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 Ca(2+)-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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aminoacyltransferases / chemistry*
  • Aminoacyltransferases / genetics
  • Aminoacyltransferases / metabolism*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Catalysis
  • Crystallography, X-Ray
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism*
  • Kinetics
  • Models, Molecular
  • Mutagenesis / genetics*
  • Mutation / genetics
  • Peptidyl Transferases / chemistry*
  • Peptidyl Transferases / genetics
  • Peptidyl Transferases / metabolism*
  • Protein Denaturation
  • Protein Structure, Tertiary
  • Staphylococcus aureus / enzymology*
  • Staphylococcus aureus / genetics
  • Substrate Specificity
  • Temperature


  • Bacterial Proteins
  • Aminoacyltransferases
  • sortase A
  • Peptidyl Transferases
  • Cysteine Endopeptidases