Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA

Biochemistry. 2004 Feb 17;43(6):1541-51. doi: 10.1021/bi035920j.


The Staphylococcus aureus sortase transpeptidase SrtA isoform is responsible for the covalent attachment of virulence and colonization-associated proteins to the bacterial peptidoglycan. SrtA utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-D-isoGlu-Lys(epsilon-Gly(5))-D-Ala-D-Ala (branched Lipid II) and secreted proteins containing a highly conserved C-terminal LPXTG sequence. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly(5) portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we describe the development of a general in vitro method for elucidating the substrate specificity of sortase enzymes. In addition, using immunofluorescence, cell adhesion assays, and transmission electron microscopy, we establish links between in vitro substrate specificity and in vivo function of the S. aureus sortase isoforms. Results from these studies provide strong supporting evidence of a primary role of the SrtA isoform in S. aureus adhesion and host colonization, illustrate a lack of specificity cross talk between SrtA and SrtB isoforms, and highlight the potential of SrtA as a target for the development of antivirulence chemotherapeutics against Gram-positive bacterial pathogens.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Aminoacyltransferases / chemistry*
  • Aminoacyltransferases / deficiency
  • Aminoacyltransferases / genetics
  • Aminoacyltransferases / ultrastructure
  • Bacterial Adhesion
  • Bacterial Proteins
  • Cell Wall / chemistry
  • Cell Wall / genetics
  • Cell Wall / ultrastructure
  • Cloning, Molecular
  • Conserved Sequence
  • Cysteine Endopeptidases
  • Enzyme Activation / genetics
  • Histidine / chemistry
  • Isoenzymes / chemistry
  • Isoenzymes / deficiency
  • Isoenzymes / genetics
  • Isoenzymes / ultrastructure
  • Mutation
  • Peptide Library
  • Staphylococcus aureus / enzymology*
  • Staphylococcus aureus / genetics
  • Staphylococcus aureus / pathogenicity
  • Staphylococcus aureus / ultrastructure
  • Substrate Specificity
  • Virulence


  • Bacterial Proteins
  • Isoenzymes
  • Peptide Library
  • Histidine
  • Aminoacyltransferases
  • sortase A
  • Cysteine Endopeptidases