Crystal Structures of Staphylococcus aureus Sortase A and Its Substrate Complex (2004)

The cell wall envelope of staphylococci and other Gram-positive pathogens is coated with surface proteins that interact with human host tissues. Surface proteins of Staphylococcus aureus are covalently linked to the cell wall envelope by a mechanism requiring C-terminal sorting signals with an LPXTG motif. Sortase (SrtA) cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between threonine at the C-terminal end of polypeptides and cell wall cross-bridges. The active site architecture and catalytic mechanism of sortase A has hitherto not been revealed. Here we present the crystal structures of native SrtA, of an active site mutant of SrtA, and of the mutant SrtA complexed with its substrate LPETG peptide and describe the substrate binding pocket of the enzyme. Highly conserved proline (P) and threonine (T) residues of the LPXTG motif are held in position by hydrophobic contacts, whereas the glutamic acid residue (E) at the X position points out into the solvent. The scissile T-G peptide bond is positioned between the active site Cys184 and Arg197 residues and at a greater distance from the imidazolium side chain of His120. All three residues, His120, Cys184, and Arg197, are conserved in sortase enzymes from Gram-positive bacteria. Comparison of the active sites of S. aureus sortase A and sortase B provides insight into substrate specificity and suggests a universal sortase-catalyzed mechanism of bacterial surface protein anchoring in Gram-positive bacteria. The cell wall envelope of staphylococci and other Gram-positive pathogens is coated with surface proteins that interact with human host tissues. Surface proteins of Staphylococcus aureus are covalently linked to the cell wall envelope by a mechanism requiring C-terminal sorting signals with an LPXTG motif. Sortase (SrtA) cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between threonine at the C-terminal end of polypeptides and cell wall cross-bridges. The active site architecture and catalytic mechanism of sortase A has hitherto not been revealed. Here we present the crystal structures of native SrtA, of an active site mutant of SrtA, and of the mutant SrtA complexed with its substrate LPETG peptide and describe the substrate binding pocket of the enzyme. Highly conserved proline (P) and threonine (T) residues of the LPXTG motif are held in position by hydrophobic contacts, whereas the glutamic acid residue (E) at the X position points out into the solvent. The scissile T-G peptide bond is positioned between the active site Cys184 and Arg197 residues and at a greater distance from the imidazolium side chain of His120. All three residues, His120, Cys184, and Arg197, are conserved in sortase enzymes from Gram-positive bacteria. Comparison of the active sites of S. aureus sortase A and sortase B provides insight into substrate specificity and suggests a universal sortase-catalyzed mechanism of bacterial surface protein anchoring in Gram-positive bacteria. Many bacteria adhere to host extracellular matrix proteins as an essential first step toward the pathogenesis of infection (1Foster T.J. Höök M. Trends Microbiol. 1998; 6: 484-488Google Scholar). In Gram-positive bacteria, the cell wall envelope serves as a surface organelle with immobilized surface proteins that are responsible for mediating adhesion to host tissues (2Navarre W.W. Schneewind O. Microbiol. Mol. Biol. Rev. 1999; 63: 174-229Google Scholar). Many of these surface proteins are covalently linked to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with the conserved LPXTG motif (where X represents any amino acid) (3Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Google Scholar). Sortase A, a transpeptidase with an active site cysteine, cleaves the sorting signal between the threonine and the glycine of the LPXTG motif (4Mazmanian S.K. Liu G. Ton-That H. Schneewind O. Science. 1999; 285: 760-763Google Scholar, 5Mazmanian S.K. Liu G. Jensen E.R. Lenoy E. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5510-5515Google Scholar, 6Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Google Scholar, 7Navarre W.W. Schneewind O. Mol. Microbiol. 1994; 14: 115-121Google Scholar). Sortase catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of the cell wall cross-bridge, a pentaglycyl moiety in staphylococci (8Schneewind O. Fowler A. Faull K.F. Science. 1995; 268: 103-106Google Scholar, 9Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1997; 272: 22285-22292Google Scholar). Several observations suggest that lipid II, a membrane anchored intermediate of cell wall synthesis, functions as the peptidoglycan substrate of sortase A (10Perry A.M. Ton-That H. Mazmanian S.K. Schneewind O. J. Biol. Chem. 2002; 277: 16241-16248Google Scholar, 11Ruzin A. Severin A. Ritacco F. Tabei K. Ingh S.G. Bradford P.A. Siegel M.M. Projan S.J. Shlaes D.M. J. Bacteriol. 2002; 184: 2141-2147Google Scholar). The product of the complete sorting reaction, surface protein tethered to lipid II, is presumed to be incorporated into the cell wall envelope via the penicillin-sensitive transpeptidation and transglycosylation reactions of peptidoglycan synthesis (10Perry A.M. Ton-That H. Mazmanian S.K. Schneewind O. J. Biol. Chem. 2002; 277: 16241-16248Google Scholar, 12Ton-That H. Labischinski H. Berger-Bächi B. Schneewind O. J. Biol. Chem. 1998; 273: 29143-29149Google Scholar). Sortase A (SrtA) is a polypeptide of 206 amino acids with an N-terminal membrane-spanning region and a C-terminal catalytic domain (4Mazmanian S.K. Liu G. Ton-That H. Schneewind O. Science. 1999; 285: 760-763Google Scholar, 14Mazmanian S.K. Ton-That H. Schneewind O. Mol. Microbiol. 2001; 40: 1049-1057Google Scholar). Mutant staphylococci harboring a deletion of the srtA gene accumulate surface protein precursor molecules with C-terminal sorting signals in the membrane compartment (5Mazmanian S.K. Liu G. Jensen E.R. Lenoy E. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5510-5515Google Scholar). Although Δ(srtA) staphylococci grow on laboratory media similar to the wild-type strain, the sortase mutants display severe defects in the pathogenesis of animal infections (16Jonsson I.M. Mazamanian S.K. Schneewind O. Vendrengh M. Bremell T. Tarkowski A. J. Infect. Dis. 2002; 185: 1417-1424Google Scholar, 17Jonsson I.M. Mazmanian S.K. Schneewind O. Bremell T. Tarkowski A. Microb. Infect. 2003; 5: 775-780Google Scholar). Genes homologous to Staphylococcus aureus srtA are found in all Gram-positive bacterial genomes (18Pallen M.J. Lam A.C. Antonio M. Dunbar K. Trends Microbiol. 2001; 9: 97-102Google Scholar). Considerable evidence has now accumulated that the inactivation of sortase genes interferes with the anchoring and the surface display of distinct sets of proteins (defined by their sorting signals), thereby reducing the adhesiveness and virulence of bacterial pathogens (19Bolken T.C. Franke C.A. Jones K.F. Zeller G.O. Jones C.H. Dutton E.K. Hruby D.E. Infect. Immun. 2001; 69: 75-80Google Scholar, 20Kharat A.S. Tomasz A. Infect. Immun. 2003; 71: 2758-2765Google Scholar, 21Hava D.L. Hemsley C.J. Camilli A. J. Bacteriol. 2003; 185: 413-421Google Scholar, 22Barnett T.C. Scott J.R. J. Bacteriol. 2002; 184: 2181-2191Google Scholar, 23Lee S.F. Boran T.L. Infect. Immun. 2003; 71: 676-681Google Scholar, 24Bierne H. Mazmanian S.K. Trost M. Pucciarelli M.G. Dehoux P. Jansch L. Garcia-del Portillo F. Schneewind O. Cossart P. Mol. Microbiol. 2002; 43: 869-881Google Scholar, 25Garandeau C. Reglier-Poupet H. Dubail I. Beretti J.L. Berche P. Charbit A. Infect. Immun. 2002; 70: 1382-1390Google Scholar, 26Ton-That H. Schneewind O. Mol. Microbiol. 2003; 50: 1429-1438Google Scholar). Because of the central role of sortases in the functional assembly of the cell wall envelope and in bacterial pathogenicity, sortases have been acknowledged as a target for the development of therapeutic agents that may disrupt human infections caused by Gram-positive bacteria (27Cossart P. Jonquieres R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5013-5015Google Scholar). A single conserved cysteine (Cys184) in SrtA is absolutely essential for its activity (6Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Google Scholar). Substitution of Cys184 with Ala or addition of cysteine reactive reagents such as methyl methane-thiosulfonate and p-hydroxymercury benzoate abolish the activity of the enzyme, suggesting that SrtA is a cysteine protease (6Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Google Scholar, 28Ton-That H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Google Scholar, 29Ton-That H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Google Scholar, 30Ton-That H. Mazmanian S.K. Alksne L. Schneewind O. J. Biol. Chem. 2002; 277: 7447-7452Google Scholar). The cleavage of the LPXTG motif between the threonine (T) and glycine (G) residues by SrtA leads to the formation of a covalent thioester bond between the thiol group of the enzyme cysteine and the carboxyl group of the substrate threonine residue, and this transient acyl-enzyme intermediate is subsequently resolved by the nucleophilic attack of the amino group of pentaglycyl (6Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Google Scholar, 13Kruger R.G. Otvos B. Frankel B.A. Bentley M. Dostal P. McCafferty D.G. Biochemistry. 2004; 43: 1541-1551Google Scholar). A new amide bond is then formed between the threonine and the N-terminal glycine residue of the peptidoglycan, resulting in a substrate protein covalently linked to the peptidoglycan (8Schneewind O. Fowler A. Faull K.F. Science. 1995; 268: 103-106Google Scholar). SrtAΔN59, a recombinant sortase A the N-terminal residues, is functional in and catalyzes the cleavage of an LPXTG motif as as the transpeptidation with substrate H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Google Scholar, U. Ton-That H. J. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar). of the of three residues, Cys184 with and in a similar to that of the catalytic in cysteine such as U. Ton-That H. J. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar). Because a is and conserved the of cysteine positioned Cys184 and residues in SrtA to as the catalytic H. Mazmanian S.K. Alksne L. Schneewind O. J. Biol. Chem. 2002; 277: 7447-7452Google Scholar, U. Ton-That H. J. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar). the thiol group of Cys184 from the of and the distance between the side that in a catalytic In the side chain of His120, a hydrophobic may not be to a A the of the active site Cys184 thiol and to be and suggesting that SrtA not a in its active site R. U. J. Biol. Chem. 2003; Scholar). the of the active site the catalytic of SrtA is In the crystal of S. aureus sortase a cysteine transpeptidase responsible for anchoring surface proteins with C-terminal sorting to the cell the of sortases a catalytic for these the of the substrate binding pocket that may S. aureus sortases to between LPETG and sorting signals Mazmanian S.K. Schneewind O. 2004; Scholar). we to the substrate binding pocket and the catalytic mechanism of sortase A by the crystal structures of SrtAΔN59, its active site mutant SrtAΔN59, we the active nucleophilic residue Cys184 to an and a of the mutant and its peptide substrate LPETG by S. aureus and sortase B crystal structures Mazmanian S.K. Schneewind O. 2004; we to the substrate binding pocket of sortases and their catalytic and a catalytic mechanism that may be universal for and recombinant enzyme and its and in as (6Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Google Scholar). The of the N-terminal amino acids not the activity of the enzyme H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Google Scholar, U. Ton-That H. J. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2001; the and the All by the with a protein of in The and of A single crystal The recombinant enzyme to be active at not The crystal and in its to be for All other by the an S. 1997; and the by with to a LPETG peptide from the of peptide synthesis The by the peptide into the similar native with a to of at on the of three and with and the of 1997; Scholar). A complete for the at the the crystal of by the single the at of and with the of and P. J. M. T. Biol. 1998; Scholar). The crystal of by Model with the of the T. M. M. S. and in and with the of The and are in and at of at at of I. of sites of B for for for of protein of at of at at of I. in a new of of and to the group and are molecules and in the in the crystal by the structures as Although on a crystal of between the three the single to the crystal by the A of sites of found the sites for P. J. M. T. Biol. 1998; Scholar). The to In addition to the crystal the crystal structures of native and the LPETG peptide by and to and The of the crystal is an and the a A the of proteins in the protein The three molecules and present in the are not by any N-terminal residues in A, in and in have in N-terminal The between molecules A, and are The the three molecules is in the of the the and in side of the formed by the and is in with three of the a hydrophobic pocket in the of the catalytic Cys184 is The side chain of Cys184 is out into the to this pocket is in the of molecules B and by the of whereas the hydrophobic pocket of the active site residue in A has from the solvent. of the crystal with of its that the to be with an of for all the observations may to the by structures as are in the side chain between the crystal and structures in the region as as in of the and these may be an of the of Comparison with SrtA from S. aureus the first cysteine transpeptidase to be (6Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Google Scholar). The in the transpeptidation the cleavage of the scissile bond of a substrate and the formation of an acyl-enzyme that are similar to in reactions by all cysteine (6Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Google Scholar, 30Ton-That H. Mazmanian S.K. Alksne L. Schneewind O. J. Biol. Chem. 2002; 277: 7447-7452Google Scholar). Comparison of SrtA with other cysteine of the mechanism of this enzyme. Cys184 and are absolutely conserved in all sortase enzymes and to be essential for SrtA H. Mazmanian S.K. Alksne L. Schneewind O. J. Biol. Chem. 2002; 277: 7447-7452Google Scholar, U. Ton-That H. J. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar). observations the for a SrtA may a for H. Mazmanian S.K. Alksne L. Schneewind O. J. Biol. Chem. 2002; 277: 7447-7452Google Scholar). A residue, in in the formation of a H. Mazmanian S.K. Alksne L. Schneewind O. J. Biol. Chem. 2002; 277: 7447-7452Google Scholar). for the the catalytic cysteine and residues are positioned on at the of a and an and are in J. J. Mol. Biol. Scholar, J. J. Mol. Biol. Scholar). The site is a between the catalytic residues, and of are into the and of the scissile amide whereas the amide of the catalytic and a residue side chain to as the and the intermediate In the crystal of SrtAΔN59, Cys184 and are anchored on of a the of the catalytic residues, and residues between sortase A, and sortase B is similar U. Ton-That H. J. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2001; Scholar, Mazmanian S.K. Schneewind O. 2004; Scholar). the thiol group of Cys184 in is positioned from the of His120, and this distance not be to the for residues of a Cys184 and not be positioned in to a for the that a may not be formed in the active site by the of the active site Cys184 thiol and to be and R. U. J. Biol. Chem. 2003; Scholar). In cysteine and the catalytic residue an role in the reactive nucleophilic attack by the amide of the substrate scissile bond M.J. J. Chem. 1997; Scholar, A.C. R. 1994; Scholar). in other residues such as and are to this role M. 1998; Scholar, Biochemistry. 2003; Scholar). of the active site Cys184 residue in the crystal in addition to His120. in a of and residues Arg197, and be the residues Arg197 and are on the Cys184 side of the whereas are into the or to the of substrate binding and a residue side chain that the catalytic residues are anchored a we a single the side chain group of Arg197, that be in of Cys184 to a catalytic M.J. J. Chem. 1997; Scholar). The of and SrtA to with in (6Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Google Scholar). The of and LPETG peptide not and the thiol group of Cys184 found in and to the complete acyl-enzyme intermediate peptide be of the of and the of as a to the peptide be of the of the peptide as sortase has been to a in in the of the peptidoglycan H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Google Scholar, A. B. Alksne L. Tabei K. G. Biochemistry. 2003; Scholar). LPETG peptide into the of an active site mutant are with of the in the of in the active site of in the such for molecules A and B. is of as the sites of A and B are by the of the The in a surface is in a region by The N-terminal of the peptide is positioned toward the of the whereas its C-terminal end to the The conserved and residues of the peptide (4Mazmanian S.K. Liu G. Ton-That H. Schneewind O. Science. 1999; 285: 760-763Google have in the hydrophobic formed by the residues present on the of the and the The side chain of the conserved residue in the LPXTG the residue of the substrate (4Mazmanian S.K. Liu G. Ton-That H. Schneewind O. Science. 1999; 285: 760-763Google is into the solvent. the scissile peptide bond between the C-terminal and residues of the peptide is positioned between and Arg197, from the side the of is toward the Arg197 group and the between for is in the as for the scissile and of the in the active M.J. J. Chem. 1997; Scholar). The binding of the peptide substrate not the of the enzyme. The side chain is at a distance of from the substrate scissile peptide A of hydrophobic residues, and to the substrate on this be out as a the that essential for activity H. Mazmanian S.K. Alksne L. Schneewind O. J. Biol. Chem. 2002; 277: 7447-7452Google suggesting that may be a role that of a catalytic on cysteine protease suggest that the of the nucleophilic cysteine is not by the of the S. M. S. A. A. H. S.K. C. K. Biochemistry. 1997; Scholar). The and that to the in the active site are for the catalytic of the S. M. S. A. A. H. S.K. C. K. Biochemistry. 1997; Scholar, A.S. M. 2003; Scholar). Several residues a of the thiol group of Cys184, of is may a role in to such an The that the and of may be for the and hydrophobic has been as of the mutant enzyme not be of to Ala may in the architecture of the active site that be responsible for the of the In the of the residue to as a catalytic is the Arg197 Arg197 is the group to the T-G bond of the substrate a the group of Arg197 as a in the the side chain of Arg197 has a and in the crystal and is in distance of the substrate peptide threonine the group to interact with the peptide bond and the and of the group may be for its role the Arg197 residue is absolutely conserved all sortases from Gram-positive bacteria Mazmanian S.K. Schneewind O. 2004; Scholar). the other the of the residue is is with that of and The of the residue a to its between a step the of Arg197 be from the for a residue J. J. Chem. as hydrophobic the of thereby a between The that Arg197 may be in the catalytic of sortases is on the side chain and to the substrate scissile and be its role in be of the Ala side chain with a thiol group for the residue the distance between the thiol group and the of the scissile peptide bond in the substrate to a that is with that in and other cysteine protease reactions M.J. J. Chem. 1997; Scholar, M.J. 1998; Scholar). In the enzyme, the thiol group of Cys184 and the group of Arg197 interact with the substrate from In in the of the is by to and present on the side of the scissile bond and the nucleophilic attack is to from In the the scissile to the the scissile bond in the cysteine protease of T. M. M. S. and in Scholar, an role for as a catalytic in sortases A. 2004; Scholar). is that the of the Cys184 thiol group in sortases may of a in of its Arg197 of the substrate amide bond the nucleophilic attack by the active Cys184 thiol and as the in the or intermediate in of the transpeptidase Comparison of the SrtA and aureus SrtA and the sorting motif and is responsible for anchoring proteins to (5Mazmanian S.K. Liu G. Jensen E.R. Lenoy E. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5510-5515Google Scholar, 6Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Google Scholar). structures of native with of in with and and substrate have been Mazmanian S.K. Schneewind O. 2004; Scholar). Although the between the enzymes is their structures are similar for the The from the and a of in the The the and in the position of the active site and are conserved H. Schneewind O. J. Biol. Chem. 1999; 274: 24316-24320Google Scholar). the chain are the catalytic and residues, with the residue the residue side chain in the in the native and in the crystal In the native crystal the side chain points from whereas the and from from that in structures suggesting a residue the for Cys184 and Arg197 are not between native and crystal be an of in catalytic and between the Although their are SrtA activity toward sorting in and the to with LPXTG sorting signals in and in (6Ton-That H. Liu G. Mazmanian S.K. Faull K.F. Schneewind O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12424-12429Google Scholar). The sorting LPETG and are homologous in and the N-terminal and C-terminal of the the that the position of the N-terminal of the LPETG peptide with the position of the of In hydrophobic residues and from the hydrophobic with the N-terminal residue of the LPETG the region in is of and with the N-terminal residue of the C-terminal end of the LPETG the active site of a residue, in this the glycine residue of the substrate in the active site of is hydrophobic in and is by residues such as and that the C-terminal end of of the of the step of the transpeptidation by the transient acyl-enzyme formed in the first is resolved by the nucleophilic attack of thereby substrate peptide from SrtA and S.K. Ton-That H. Schneewind O. Mol. Microbiol. 2001; 40: 1049-1057Google Scholar). on the crystal of the Mazmanian S.K. Schneewind O. 2004; we a for a The N-terminal of be positioned in to catalytic residues Cys184 and Arg197 and the peptide may be by the region between and a conserved is found in to the catalytic in all three molecules in the of SrtAΔN59, or the held by to the of the and is from the thiol group of Cys184 and from the of the group of that this is positioned to be by the substrate the of the transpeptidation A similar found in the active site of crystal structures and is by the substrate in the crystal of and the Mazmanian S.K. Schneewind O. 2004; Scholar). that this conserved the of the peptide substrate in the of a in In a Mazmanian S.K. Schneewind O. 2004; Scholar, A. 2004; we S. aureus crystal structures and the that bacterial cysteine responsible for covalently surface proteins to the bacterial cell a and catalytic for the crystal structures of covalently enzyme may not the catalytic or the site of the enzyme. a with all other cysteine is to that S. aureus SrtA and sorting LPETG and In this we have the sortases may their by the structures of enzyme substrate the of the LPETG peptide crystal we the evidence for the the of the S. aureus sortase A by R. U. J. Biol. Chem. 2003; and that the conserved residue in the active site of sortases is not positioned to the role in a peptide In the crystal structures of LPETG the from the Mazmanian S.K. Schneewind O. 2004; Scholar, A. 2004; a role for the conserved residue in the of the on S. aureus sortase A suggest that the enzyme functions as a in the of nucleophilic peptidoglycan or its H. Mazmanian H. Faull K.F. Schneewind O. J. Biol. Chem. 2000; 275: 9876-9881Google Scholar, A. B. Alksne L. Tabei K. G. Biochemistry. 2003; Scholar). such a of is conserved in the of the catalytic residues and in the crystal structures of SrtA and has been that sortases the transpeptidation a the substrate to the enzyme the acyl-enzyme is formed A. B. Alksne L. Tabei K. G. Biochemistry. 2003; Scholar). is by that the is by the substrate is into the of the and by to for into the native and The that the amide bond between the first and glycine in the substrate is essential for binding to the enzyme active site in the crystal the that the catalytic and residues has or with peptide Mazmanian S.K. Schneewind O. 2004; Scholar). The for the LPETG peptide binding to SrtA from to for the of the peptide in crystal S. aureus sortases SrtA and with and conserved of catalytic in the catalytic the of residues to residues in SrtAΔN59, the for the peptide substrate in the region of SrtAΔN59, by three residues the of from the catalytic site of the enzyme and and the site in the crystal this we that the site of is of the position and toward the The of and residues into the substrate binding pocket the of the substrate binding the position and of a of hydrophobic residues, and at the of the substrate binding pocket of SrtA, the of the hydrophobic and residues of the LPETG peptide In addition to and residues, and residues of into its substrate binding by hydrophobic and residues in SrtA be specificity toward sorting