Anchor Structure of Staphylococcal Surface Proteins

Surface proteins of Staphylococcus aureus are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LP X TG motif. Cleavage between the threonine and the glycine of the LP X TG motif liberates the carboxyl of threonine to form an amide bond with the amino of the pentaglycine cross-bridge in the staphylococcal peptidoglycan. We asked whether antibiotic cell wall synthesis inhibitors interfere with the anchoring of surface proteins. Penicillin G, a transpeptidation inhibitor, had no effect on surface protein anchoring, whereas vancomycin and moenomycin, inhibitors of cell wall polymerization into peptidoglycan strands, slowed the sorting reaction. Cleavage of surface protein precursors did not require a mature assembled cell wall and was observed in staphylococcal protoplasts. A search for chemical inhibitors of the sorting reaction identified methanethiosulfonates and p- hydroxymercuribenzoic acid. Thus, sortase, the enzyme proposed to cleave surface proteins at the LP X TG motif, appears to be a sulfhydryl-containing enzyme that utilizes peptidoglycan precursors but not an assembled cell wall as a substrate for the anchoring of surface protein. Surface proteins of Staphylococcus aureus are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LP X TG motif. Cleavage between the threonine and the glycine of the LP X TG motif liberates the carboxyl of threonine to form an amide bond with the amino of the pentaglycine cross-bridge in the staphylococcal peptidoglycan. We asked whether antibiotic cell wall synthesis inhibitors interfere with the anchoring of surface proteins. Penicillin G, a transpeptidation inhibitor, had no effect on surface protein anchoring, whereas vancomycin and moenomycin, inhibitors of cell wall polymerization into peptidoglycan strands, slowed the sorting reaction. Cleavage of surface protein precursors did not require a mature assembled cell wall and was observed in staphylococcal protoplasts. A search for chemical inhibitors of the sorting reaction identified methanethiosulfonates and p- hydroxymercuribenzoic acid. Thus, sortase, the enzyme proposed to cleave surface proteins at the LP X TG motif, appears to be a sulfhydryl-containing enzyme that utilizes peptidoglycan precursors but not an assembled cell wall as a substrate for the anchoring of surface protein. staphylococcal enterotoxin B staphyloccal protein A staphylococcal β-lactamase chemically defined medium cell wall sorting signal N-acetylglucosamine N-acetylmuramic acid polyacrylamide gel electrophoresis 2-(trimethylammonium)ethylmethanethiosulfonate To infect and multiply within their human hosts, Gram-positive bacteria require surface proteins that either bind to specific organ tissues or provide ingenious strategies for bacterial escape from the immune response (1Navarre W.W. Schneewind O. Microbiol. Mol. Biol. Rev. 1999; 63: 174-229Crossref PubMed Google Scholar). The mechanism of surface protein anchoring to the bacterial cell wall has recently been established for protein A of Staphylococcus aureus. After synthesis in the cytoplasm, protein A is initiated into the secretory pathway by an NH2-terminal leader peptide (2Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Abstract Full Text PDF PubMed Scopus (438) Google Scholar). A COOH-terminal sorting signal is necessary and sufficient for the anchoring of protein A and functions first to retain the polypeptide within the secretory pathway (3Schneewind O. Mihaylova-Petkov D. Model P. EMBO J. 1993; 12: 4803-4811Crossref PubMed Scopus (360) Google Scholar, 4Navarre W.W. Schneewind O. J. Bacteriol. 1996; 178: 441-446Crossref PubMed Google Scholar); this allows proteolytic cleavage between the threonine and the glycine of a conserved LP X TG motif (5Navarre W.W. Schneewind O. Mol. Microbiol. 1994; 14: 115-121Crossref PubMed Scopus (310) Google Scholar). The carboxyl of threonine is amide-linked to the amino of the pentaglycine cross-bridge (6Schneewind O. Fowler A. Faull K.F. Science. 1995; 268: 103-106Crossref PubMed Scopus (375) Google Scholar), thereby tethering the COOH-terminal end of protein A to the staphylococcal peptidoglycan (7Navarre W.W. Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1998; 273: 29135-29142Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 8Navarre W.W. Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1999; 274: 15847-15856Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 9Ton-That H. Faull K.F. Schneewind O. J. Biol. Chem. 1997; 272: 22285-22292Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 10Ton-That H. Labischinski H. Berger-Bächi B. Schneewind O. J. Biol. Chem. 1998; 273: 29143-29149Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This amide bond exchange mechanism displays a striking similarity to the penicillin-sensitive transpeptidation reaction, during which murein pentapeptide is cleaved between d-Ala-d-Ala and the liberated carboxyl of d-Ala at position four is amide-linked to the amino of the pentaglycine cross-bridge (11Tipper D.J. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1133-1141Crossref PubMed Scopus (637) Google Scholar). Elements involved in transpeptidation and cell wall sorting are conserved, suggesting that surface protein anchoring is a universal mechanism in Gram-positive pathogens (1Navarre W.W. Schneewind O. Microbiol. Mol. Biol. Rev. 1999; 63: 174-229Crossref PubMed Google Scholar, 12Schleifer K.H. Kandler O. Bacteriol. Rev. 1972; 36: 407-477Crossref PubMed Google Scholar). If so, sortase, the enzymatic activity that is thought to catalyze this reaction, might also be found conserved in many different bacterial species. Methods that inhibit sortase may be used as anti-bacterial therapies for the treatment of human infections caused by Gram-positive microbes. Although surface protein anchoring has been characterized in molecular detail, little is known about the peptidoglycan substrate. Because a biochemical in vitro reaction for sortase has not yet been established, we approached this question by searching for inhibitors of cell wall sorting. During bacterial cell wall synthesis, a soluble cytoplasmic peptidoglycan precursor (UDP-MurNac-l-Ala-d-iGln-l-Lys-d-Ala-d-Ala, where iGln is iso-glutaminyl; Park's nucleotide) is linked to undecaprenolpyrophosphate to generate lipid I (13Chatterjee A.N. Park J.T. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 9-16Crossref PubMed Scopus (43) Google Scholar, 14Matsuhashi M. Dietrich C.P. Strominger J.L. J. Biol. Chem. 1967; 242: 3191-3206Abstract Full Text PDF Google Scholar, 15Matsuhashi M. Ghuysen J.-M. Hakenbeck R. Bacterial Cell Wall. Elsevier Biochemical Press, Amsterdam1994: 55-72Google Scholar). This membrane-bound intermediate is further modified by the addition of GlcNac and pentaglycine (undecaprenolpyrophosphate-MurNac(-l-Ala-d-iGln-(NH2-Gly5)-l-Lys-d-Ala-d-Ala)-(β1–4)-GlcNac; also named lipid II) and then translocated across the cytoplasmic membrane (14Matsuhashi M. Dietrich C.P. Strominger J.L. J. Biol. Chem. 1967; 242: 3191-3206Abstract Full Text PDF Google Scholar, 16Petit J.-F. Strominger J.L. Söll D. J. Biol. Chem. 1968; 243: 757-767Abstract Full Text PDF PubMed Google Scholar, 17Matsuhashi M. Dietrich C.P. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 587-594Crossref PubMed Scopus (66) Google Scholar). Lipid II serves as a substrate for the transglycosylation reaction, which polymerizes the glycan strands of the bacterial cell wall to yield repeating disaccharide (MurNac-GlcNac)n (18Nakagawa J. Tamaki S. Tomioka S. Matsuhashi M. J. Biol. Chem. 1984; 259: 13937-13946Abstract Full Text PDF PubMed Google Scholar). Cell wall pentapeptides (l-Ala-d-iGln-l-Lys-d-Ala-d-Ala) linked to the MurNac of nascent peptidoglycan strands are cross-linked via the transpeptidation reaction, thereby generating a three-dimensional cell wall network that protects bacteria from osmotic lysis (11Tipper D.J. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1133-1141Crossref PubMed Scopus (637) Google Scholar, 19Izaki K. Matsuhashi M. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 656-663Crossref PubMed Scopus (110) Google Scholar). Penicillin is an inhibitor of the transpeptidation reaction (20Strominger J.L. Izaki K. Matsuhashi M. Tipper D.J. Fed. Proc. 1967; 26: 9-18PubMed Google Scholar). This class of β-lactam antibiotics functions as a molecular mimicry ofd-Ala-d-Ala (11Tipper D.J. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1133-1141Crossref PubMed Scopus (637) Google Scholar) and, after cleavage of the antibiotic by transpeptidases (penicillin-binding proteins), continues to occupy their active site serine (21Yocum R.R. Waxman D.J. Rasmussen J.R. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2730-2734Crossref PubMed Scopus (94) Google Scholar). Although penicillin effectively blocks the cross-linking of wall peptides (19Izaki K. Matsuhashi M. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 656-663Crossref PubMed Scopus (110) Google Scholar), it does not interfere with the transglycosylation reaction (22Tipper D.J. Strominger J.L. J. Biol. Chem. 1968; 243: 3169-3179Abstract Full Text PDF PubMed Google Scholar). Vancomycin binds to the d-Ala-d-Ala moiety of lipid II (23Bugg T.D.H. Wright G.D. Dutka-Malen S. Arthur M. Courvalin P. Walsh C.T. Biochemistry. 1991; 30: 10408-10415Crossref PubMed Scopus (523) Google Scholar, 24Handwerger S. Pucci M.J. Volk K.J. Liu J. Lee M.S. J. Bacteriol. 1992; 174: 5982-5984Crossref PubMed Google Scholar), thereby preventing substrate recognition by penicillin-binding proteins that catalyze both transglycosylase and transpeptidase reactions (11Tipper D.J. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1965; 54: 1133-1141Crossref PubMed Scopus (637) Google Scholar,25Tipper D.J. Biochemistry. 1968; 7: 1441-1449Crossref Scopus (72) Google Scholar). Moenomycin is an inhibitor of transglycosylation because this compound inhibits C55-isoprenoid-alcohol kinase (26Sandermann H. Biochim. Biophys. Acta. 1976; 444: 783-788Crossref PubMed Scopus (5) Google Scholar, 27Sandermann H. Strominger J.L. J. Biol. Chem. 1972; 247: 5123-5131Abstract Full Text PDF PubMed Google Scholar) as well as the transglycosylase activity of penicillin-binding proteins (28van Heijenoort Y. Leduc M. Singer H. van Heijenoort J. J. Gen. Microbiol. 1987; 133: 667-674PubMed Google Scholar). Here we report that vancomycin and moenomycin, but not penicillin, cause a reduction in the rate of surface protein anchoring, suggesting that sortase may utilize lipid II as a substrate for the sorting reaction. Consistent with this hypothesis is our observation that staphylococcal protoplasts, in which the assembled cell wall has been removed by digestion with muralytic enzyme, catalyze the cleavage of surface proteins at their LP X TG motif similar to bacteria with an intact cell wall. A search for chemical inhibitors of the sorting reaction identified methanethiosulfonates as well as organic mercurials, indicating that sortase must be a sulfhydryl-containing enzyme. Plasmids pSeb-Spa490–524 (3Schneewind O. Mihaylova-Petkov D. Model P. EMBO J. 1993; 12: 4803-4811Crossref PubMed Scopus (360) Google Scholar), pSeb-Cws-BlaZ, and pSeb-CwsΔLP X TG-BlaZ (5Navarre W.W. Schneewind O. Mol. Microbiol. 1994; 14: 115-121Crossref PubMed Scopus (310) Google Scholar) were transformed into S. aureus OS2 (spa:ermC, r −) (2Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Abstract Full Text PDF PubMed Scopus (438) Google Scholar) and have been described previously. Staphylococci were generally grown in tryptic soy broth or agar. All chemicals were purchased from Sigma unless indicated otherwise. S. aureus OS2(pSeb-Spa490–524) was grown overnight in CDM (29van de Rijn I. Kessler R.E. Infect. Immun. 1980; 27: 444-448Crossref PubMed Google Scholar) (Jeol BioSciences) supplemented with chloramphenicol (10 μg/ml), diluted 1:10 into minimal medium, and grown with shaking at 37 °C until A600 0.6. Cells were labeled with 100 μCi of35S-labeled Promix (Amersham Pharmacia Biotech) for 1 min. Labeling was quenched by the addition of an excess nonradioactive amino acid (50-μl chase (100 mg/ml casamino acids, 20 mg/ml methionine and cysteine)). At timed intervals (0, 1, 3, and 10 min) after the addition of the chase, 250-μl aliquots were removed, and proteins were precipitated by the addition of 250 μl of 10% trichloroacetic acid. The precipitate was sedimented by centrifugation at 15,000 ×g for 10 min, washed with 1 ml of acetone, and dried. Samples were suspended in 1 ml of 0.5 m Tris-HCl, pH 6.8, and staphylococcal peptidoglycan was digested by adding 50 μl of lysostaphin (30Schindler C.A. Schuhardt V.T. Proc. Natl. Acad. Sci. U. S. A. 1964; 51: 414-421Crossref PubMed Scopus (323) Google Scholar) (100 μg, AMBI Pharmaceuticals) and incubating for 1 h at 37 °C. Proteins were again precipitated with trichloroacetic acid, washed with acetone, and subjected to immunoprecipitation with α-Seb1 followed by SDS-PAGE and PhosphorImager analysis. To characterize the P1 and P2 precursors, 1 ml of culture was either incubated with 5 mm sodium azide for 5 to or 5 mm was after the of the of S. aureus OS2(pSeb-Spa490–524) grown in CDM were diluted into minimal medium and incubated until A600 were then with penicillin (10 μg/ml), vancomycin (10 μg/ml), or (10 or were A culture was removed for with 100 μCi of Promix for 5 min. Labeling was quenched and proteins precipitated by the addition of 0.5 ml of 10% trichloroacetic acid. The precipitate was by washed in acetone, and The were suspended in 1 ml of 0.5 m Tris-HCl, pH 50 μl of lysostaphin (100 AMBI Pharmaceuticals) was and the staphylococcal cell wall was digested by incubating for 1 h at 37 °C. Proteins were precipitated with trichloroacetic acid, washed in acetone, in 50 μl of 0.5 pH and for 10 min. of surface protein were with followed by SDS-PAGE and PhosphorImager analysis. Staphylococci were grown in the or of antibiotics as described At culture were and labeled with either 50 μCi of or 50 μCi of for 20 D. G.D. PubMed Scopus Google Scholar). All was quenched by the addition of 0.5 ml of trichloroacetic acid. Samples were to °C for min, to and The were into a and washed with ml of and ml of 50 mm Tris-HCl, pH After in 5 ml of mm Tris-HCl, pH 1 mg/ml at °C for min, were washed again with ml of and ml of The of by the was by D. G.D. PubMed Scopus Google Scholar). S. aureus OS2(pSeb-Spa490–524) was grown overnight in CDM supplemented with chloramphenicol (10 μg/ml), diluted 1:10 into minimal medium, and grown with shaking at 37 °C until A600 0.6. Cells were labeled with 100 μCi of35S-labeled Promix for 5 min. were to a of 5 mm after the of the All was quenched by adding trichloroacetic acid to and proteins were by washed in and the staphylococcal cell wall digested with lysostaphin as described The were again precipitated with trichloroacetic acid, with followed by SDS-PAGE and PhosphorImager analysis. of S. aureus or S. aureus X grown in CDM were diluted 1:10 into minimal medium and grown with shaking at 37 °C until A600 0.6. ml of culture was with 100 μCi of Promix for min, and was quenched by the addition of 50 μl of chase aliquots were removed for trichloroacetic acid either during the or 20 after the addition of culture was first to and then subjected to The were sedimented by centrifugation at 15,000 for 5 and suspended in 1 ml of 50 mm Tris-HCl, m 10 mm pH The cell wall was digested with lysostaphin (100 for at 37 °C. The were washed in and labeled with 100 μCi of35S-labeled Promix for min, and was quenched by the addition of 50 μl of chase were at 15,000 ×g for 10 to soluble surface proteins from that were to cell All were precipitated with trichloroacetic acid, washed in acetone, and suspended in 50 μl of 0.5 m Tris-HCl, pH with for 10 min. of surface protein precursor and were with and and subjected to SDS-PAGE and PhosphorImager analysis. We to that with the anchoring of surface proteins by known inhibitors of the protein in S. aureus OS2 is as a precursor in the and initiated into the secretory pathway by an NH2-terminal leader peptide (3Schneewind O. Mihaylova-Petkov D. Model P. EMBO J. 1993; 12: 4803-4811Crossref PubMed Scopus (360) Google Scholar). After signal peptide the P2 precursor a COOH-terminal sorting signal serves as a substrate for sortase, an enzyme that between the threonine and the glycine of the LP X TG motif (5Navarre W.W. Schneewind O. Mol. Microbiol. 1994; 14: 115-121Crossref PubMed Scopus (310) Google Scholar). The liberated carboxyl of threonine is amide-linked to the amino of cell wall thereby generating surface protein (6Schneewind O. Fowler A. Faull K.F. Science. 1995; 268: 103-106Crossref PubMed Scopus (375) Google Scholar). Surface protein was by with During the P1 and P2 precursors as well as mature were 1 after the addition of the chase, surface protein was to the species. Surface protein anchoring was after the of azide is an inhibitor of an of the secretory pathway in bacteria Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of 5 mm sodium azide to staphylococcal 5 to protein and to the of a leader peptide P1 precursor (2Schneewind O. Model P. Fischetti V.A. Cell. 1992; 70: 267-281Abstract Full Text PDF PubMed Scopus (438) Google with A. Biochemistry. 1995; PubMed Scopus Google Scholar), and of 2-(trimethylammonium)ethylmethanethiosulfonate of by after the of the with the cleavage of sorting at the LP X TG motif, whereas the of the P1 precursor This that sortase must a that is necessary for enzymatic cleavage at the LP X sorting If sortase for enzymatic the addition of may also inhibit the cleavage of sorting at the LP X TG motif. This was and also with not as effectively as acid, an organic known to inhibit an whereas as and did not H. and Scholar). and did not the sorting reaction. which with H. and Scholar), treatment with the with cell wall indicating that sortase does not require or for cleavage and anchoring of surface of the sorting reaction by methanethiosulfonates and organic an of The is indicated by sorting reaction was as the between the of P2 precursor and the at the LP X TG motif an of The is indicated by in a The sorting reaction was as the between the of P2 precursor and the at the LP X TG motif To the effect of known antibiotics on cell wall we penicillin, and S. aureus OS2(pSeb-Spa490–524) was grown in minimal medium to A600 with 10 penicillin, or moenomycin, and incubated for an 5 h At intervals during this aliquots were for of surface protein sorting and cell wall The effect of antibiotics on the rate of bacterial cell wall synthesis was as the of into peptidoglycan D. G.D. PubMed Scopus Google Scholar). is a of whereas is the of of amino is a of cell wall Surface protein anchoring was by and as the between the of P2 precursor and The addition of penicillin, or the rate of as with a the rate of cell wall sorting precursor cleavage during the of the addition of vancomycin to a of P2 indicating that this compound caused a reduction of the sorting reaction. A effect was observed was to staphylococcal penicillin did not the rate of cell wall sorting. antibiotics the rate of peptidoglycan synthesis that vancomycin and cause a reduction in the rate of cell wall whereas penicillin has no effect on surface protein of cell wall synthesis and the effect on cell wall an of is indicated by of cell wall were from grown for in the of cell wall sorting reaction was as the between the of precursor and the at the LP X TG motif Cell wall synthesis was as the between the of and that of into the peptidoglycan. The are as an of is indicated by were from grown for in the of in a The cell wall sorting reaction was as the between the of precursor and the at the LP X TG motif Cell wall synthesis was as the between the of and that of into the peptidoglycan. The are as that protoplasts, by muralytic digestion of or penicillin of surface protein into the medium de Rijn I. Fischetti V.A. Infect. Immun. PubMed Google Scholar, J. J. 1976; PubMed Scopus Google Scholar). This observation be in the COOH-terminal sorting retain surface proteins in the of protoplasts, or the of an assembled cell wall is not to cleave sorting at their LP X TG motif. To between we surface protein anchoring in intact bacteria and staphylococcal cleaved the precursor to generate the NH2-terminal and COOH-terminal cytoplasmic (5Navarre W.W. Schneewind O. Mol. Microbiol. 1994; 14: 115-121Crossref PubMed Scopus (310) Google Scholar). in staphylococcal by lysostaphin digestion of the cell precursor cleavage similar to that in indicating that the of a assembled cell wall is not for cleavage of sorting We observed sorting in that in were with but not with not of the sorting reaction. The COOH-terminal of the must be from that by lysostaphin digestion of to the carboxyl of because the proteins on SDS-PAGE To whether cleaved were into the medium, were sedimented by centrifugation and from the medium in the All precursor and COOH-terminal cleavage sedimented with the protoplasts. NH2-terminal that at the as by lysostaphin digestion from the cell wall of intact were soluble in the culture but not of the sedimented suggesting that of the sorting reaction may be to precursor cleavage was observed for X TG-BlaZ either in or staphylococcal protoplasts. To characterize the peptidoglycan substrate of the cell wall sorting reaction, we If cell wall sorting the pentaglycine of assembled cell wall as a we that the treatment of bacterial with antibiotics not the anchoring of surface proteins. both and vancomycin treatment of slowed the sorting reaction. Moenomycin is an inhibitor of whereas vancomycin binds within lipid and vancomycin are known to of lipid II precursors into peptidoglycan (26Sandermann H. Biochim. Biophys. Acta. 1976; 444: 783-788Crossref PubMed Scopus (5) Google Scholar, C.T. Science. 1993; PubMed Scopus Google Scholar). of cell wall sorting was with of in the of suggesting that and vancomycin not interfere with the sorting reaction. the of peptidoglycan precursor that may as a substrate for the sorting reaction (22Tipper D.J. Strominger J.L. J. Biol. Chem. 1968; 243: 3169-3179Abstract Full Text PDF PubMed Google Scholar). If so, penicillin G, which inhibits the cross-linking of peptidoglycan strands the of lipid II (22Tipper D.J. Strominger J.L. J. Biol. Chem. 1968; 243: 3169-3179Abstract Full Text PDF PubMed Google Scholar), not the sorting reaction. This was Thus, we that the observed of surface protein anchoring by and vancomycin may be the of the of lipid II for the sorting reaction. The question of antibiotic of anchoring protein A to the cell wall has been previously. Although the mechanism of cell wall sorting was at the J. J. PubMed Scopus Google Scholar) the effect of vancomycin on the of protein A into cell wall and observed no to our of the rate of cell wall sorting precursor on the of cell protein A to as a of the of surface protein We it is that the between and our may be to the of the to cell wall anchoring of protein A. If sortase, the enzyme that cleavage of surface proteins at the LP X TG motif and to peptidoglycan does not require assembled cell cleavage of precursors in staphylococcal protoplasts. This was we also observed cleaved surface protein with on of to with the an observation that is with the of surface protein to peptidoglycan precursor H. Strominger J.L. J. Biol. Chem. 1972; 247: 5123-5131Abstract Full Text PDF PubMed Google Scholar). At this we provide biochemical for the of sorting because have not yet been and functions as a and of the sorting signal by cleavage between the threonine and the glycine of the LP X TG motif has been observed (1Navarre W.W. Schneewind O. Microbiol. Mol. Biol. Rev. 1999; 63: 174-229Crossref PubMed Google Scholar). be via an active site or of sortase, which may thereby surface proteins as enzyme J.R. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). To between the of an active site or we for inhibitors of the sorting reaction and report that cleavage at the LP X TG motif of surface proteins is to methanethiosulfonates and acid. Thus, it appears that sortase utilizes a to form an intermediate with cleaved polypeptide This hypothesis is by not interfere with with the that the active site of sortase is generally as an enzyme. of the for sortase the of a that is conserved in identified by in the of Gram-positive S. and O. in S. K. K. and O. for and Fischetti Fischetti V.A. J. PubMed Scopus Google Scholar) described a membrane cleavage enzyme activity of protein was with muralytic enzyme. The chemical of this protein cleavage and into the of is not Although membrane cleavage enzyme be by organic as acid, this activity is to the addition of Fischetti V.A. J. PubMed Scopus Google Scholar). Thus, membrane cleavage enzyme must be from sortase, as this enzyme be by either or We and for and suggesting the of We are to for the of We Model and as well as of our for this