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W2011719326 abstract "The tripeptide intermediated-Phe-Pro-Val in the biosynthesis of gramicidin S was labeled by incorporation of eitherl-[14C]phenylalanine orl-[14C]valine in an in vitrobiosynthetic assay. The gramicidin S synthetase 2-tripeptide complex was first digested with CNBr and subsequently by Staphylococcus aureus V8 protease. The active site peptide carrying the radioactively labeled tripeptide was isolated in pure form by reversed phase high performance liquid chromatography technology and analyzed by liquid phase sequencing, mass spectrometry, and amino acid analysis. It was demonstrated that d-Phe-Pro-Val is attached to the 4′-phosphopantetheine cofactor at the thiolation center for valine of gramicidin S synthetase 2. In this way the attachment site of a peptide intermediate in nonribosomal peptide biosynthesis was identified for the first time. Our results are in full agreement with the multiple carrier model of nonribosomal peptide biosynthesis (Stein, T., Vater, J., Kruft, V., Otto, A., Wittmann-Liebold, B., Franke, P., Panico, M., McDowell, R., and Morris, H. R. (1996) J. Biol. Chem. 271, 15426–15435), which predicts that the growing peptide chain in the elongation process should always be bound to the thiotemplate site specific for its C-terminal amino acid component. The tripeptide intermediated-Phe-Pro-Val in the biosynthesis of gramicidin S was labeled by incorporation of eitherl-[14C]phenylalanine orl-[14C]valine in an in vitrobiosynthetic assay. The gramicidin S synthetase 2-tripeptide complex was first digested with CNBr and subsequently by Staphylococcus aureus V8 protease. The active site peptide carrying the radioactively labeled tripeptide was isolated in pure form by reversed phase high performance liquid chromatography technology and analyzed by liquid phase sequencing, mass spectrometry, and amino acid analysis. It was demonstrated that d-Phe-Pro-Val is attached to the 4′-phosphopantetheine cofactor at the thiolation center for valine of gramicidin S synthetase 2. In this way the attachment site of a peptide intermediate in nonribosomal peptide biosynthesis was identified for the first time. Our results are in full agreement with the multiple carrier model of nonribosomal peptide biosynthesis (Stein, T., Vater, J., Kruft, V., Otto, A., Wittmann-Liebold, B., Franke, P., Panico, M., McDowell, R., and Morris, H. R. (1996) J. Biol. Chem. 271, 15426–15435), which predicts that the growing peptide chain in the elongation process should always be bound to the thiotemplate site specific for its C-terminal amino acid component. Multifunctional peptide synthetases like gramicidin S synthetase from Bacillus brevis ATCC 9999 catalyze the biosynthesis of their peptide products by a thiotemplate mechanism (1Lipmann F. Acc. Chem. Res. 1973; 6: 361-367Crossref Scopus (99) Google Scholar, 2Laland S.G. Zimmer T.-L. Essays Biochem. 1973; 9: 31-57PubMed Google Scholar, 3Kurahashi K. Annu. Rev. Biochem. 1974; 43: 445-459Crossref PubMed Scopus (59) Google Scholar, 4Laland S.G. Frøyshov Ø. Gilhuus-Moe C. Zimmer T.-L. Nat. New Biol. 1972; 239: 43-44Crossref PubMed Scopus (30) Google Scholar, 5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar). They activate their amino acid substrates in a two-step process involving aminoacyl adenylate formation and subsequent thioesterification at specific thiol groups (thiotemplates). Such multienzymes show a modular organization (6Kleinkauf H. von Döhren H. Eur. J. Biochem. 1996; 236: 335-351Crossref PubMed Scopus (283) Google Scholar, 7Marahiel M.A. Stachelhaus T. Mootz H.D. Chem. Rev. 1997; 97: 2651-2673Crossref PubMed Scopus (898) Google Scholar, 8von Döhren H. Keller U. Vater J. Zocher R. Chem. Rev. 1997; 97: 2675-2705Crossref PubMed Scopus (202) Google Scholar, 9Vater J. Stein T. Vollenbroich D. Kruft V. Wittmann-Liebold B. Franke P. Liu L. Zuber P. J. Protein Chem. 1997; 16: 557-564Crossref PubMed Scopus (23) Google Scholar). They are composed of homologous biosynthetic units (modules) consisting of 1000–1500 amino acid residues. Their arrangement along the multifunctional polypeptide chain is usually colinear to the sequence of the amino acid components in the peptide product. Such modules can be subdivided into three main domains responsible (a) for amino acid recognition, binding, and the primary activation as aminoacyl adenylates, (b) for the thiolation of the amino acid substrates, and (c) for peptide elongation, in some cases in concert with amino acid epimerization.Recently we demonstrated by affinity labeling of gramicidin S synthetase at its thiolation centers and analysis of isolated active site peptides that each module of this multienzyme is equipped with its own 4′-phosphopantetheine (4′-PPan) 1The abbreviations used are: PPan, phosphopantetheine; GS1, phenylalanine racemase, EC 5.1.1.11(gramicidin S synthetase 1); GS2, gramicidin S synthetase 2; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization. 1The abbreviations used are: PPan, phosphopantetheine; GS1, phenylalanine racemase, EC 5.1.1.11(gramicidin S synthetase 1); GS2, gramicidin S synthetase 2; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization. cofactor (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar). The sulfhydryl group of the cofactors cysteamine component functions as the thiotemplate site for the specific amino acid substrate. Each of these mobile 4′-PPan carriers is connected in a phosphodiester linkage with a specific serine that is part of a strictly conserved consensus motif LGG(H/D)S(L/I). On the basis of these results we developed a multiple carrier model of nonribosomal peptide biosynthesis (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar). This new concept replaces the old version of the thiotemplate mechanism, which was proposed by several groups in the early seventies (1Lipmann F. Acc. Chem. Res. 1973; 6: 361-367Crossref Scopus (99) Google Scholar, 2Laland S.G. Zimmer T.-L. Essays Biochem. 1973; 9: 31-57PubMed Google Scholar, 3Kurahashi K. Annu. Rev. Biochem. 1974; 43: 445-459Crossref PubMed Scopus (59) Google Scholar, 4Laland S.G. Frøyshov Ø. Gilhuus-Moe C. Zimmer T.-L. Nat. New Biol. 1972; 239: 43-44Crossref PubMed Scopus (30) Google Scholar). The old model postulated a single central 4′-PPan carrier for peptide elongation that was assumed to interact during the elongation cycle with peripheral thiols charged with the amino acid substrates in thioester linkage.According to our new concept the growing peptide chain is assembled in a series of transpeptidation reactions by successive unidirectional interaction of the individual 4′-PPan carriers thioesterified with substrates or peptide intermediates. After transpeptidation the elongated peptide chain should be attached to the 4′-PPan cofactor specific for the binding of the C-terminal amino acid. This means that in the case of gramicidin S synthetase, for example, the di-, tri-, and tetrapeptide intermediate would be bound as thioesters to the 4′-PPan cofactor at the proline, valine, and ornithine reaction centers, respectively. Unfortunately, thed-Phe-Pro-dipeptide and thed-Phe-Pro-Val-Orn-tetrapeptide complexes of gramicidin S synthetase 2 (GS2) are unstable because of internal cyclization reactions (10von Döhren H. Kleinkauf H. von Döhren H. Peptide Antibiotics: Biosynthesis and Functions. de Gruyter, Berlin1982: 169-182Google Scholar, 11Vater J. Kleinkauf H. von Döhren H. Biochemistry of Peptide Antibiotics. de Gruyter, Berlin1990: 33-55Crossref Google Scholar, 12Vater J. Schlumbohm W. Palacz Z. Salnikow J. Gadow A. Kleinkauf H. Eur. J. Biochem. 1987; 163: 297-302Crossref PubMed Scopus (17) Google Scholar). On the other hand the GS2-d-Phe-Pro-Val-tripeptide intermediate shows an exceptionally high stability (13Gadow A. Vater J. Schlumbohm W. Palacz Z. Salnikow J. Kleinkauf H. Eur. J. Biochem. 1983; 132: 229-234Crossref PubMed Scopus (18) Google Scholar). Therefore, this species is optimally qualified to solve this question. In this communication we have identified the d-Phe-Pro-Val-attachment site of gramicidin S synthetase 2. This research is of general interest to understand the mechanism of nonribosomal peptide biosynthesis on the molecular level.RESULTS AND DISCUSSIONAs demonstrated in our previous studies each amino acid activating module of a peptide synthetase using the thiotemplate mechanism is equipped with its own 4′-PPan cofactor that functions as the thiolation site for the cognate amino acids substrate (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar, 19Stein T. Vater J. Kruft V. Wittmann-Liebold B. Franke P. Panico Maria Mc Dowell R. Morris H.R. FEBS Lett. 1994; 340: 39-44Crossref PubMed Scopus (58) Google Scholar). On the basis of these results a multiple carrier model was proposed that claims that the growing peptide chain is assembled during the elongation cycle in a series of transpeptidation steps. Each peptide intermediate should be bound to the 4′-PPan carrier of that module, which is responsible for the thiolation of the C-terminal amino acid component. To verify these predictions we investigated the binding site of GS2 for the tripeptided-phenylalanyl-prolyl-valine. This intermediate is well suited for such experiments, because of the high stability of its thioester bond with the corresponding reaction center of GS2 (13Gadow A. Vater J. Schlumbohm W. Palacz Z. Salnikow J. Kleinkauf H. Eur. J. Biochem. 1983; 132: 229-234Crossref PubMed Scopus (18) Google Scholar). For this purpose we labeled gramicidin S synthetase with the radioactive tripeptide as outlined under “Experimental Procedures” using eitherl-[14C]phenylalanine orl-[14C]valine as tracer.Tripeptide formation was monitored by TLC on silica gel DC 60 plates atR f = 0.72 with butanol:acetic acid:water (4:1:1) as the mobile phase (Fig. 1). Control samples contained the same reaction mixture from whichl-proline was omitted. Under these conditions phenylalanine and valine were incorporated into GS as thioesters. After cleavage with alkali, they were detected in free form on the TLC plates instead of the tripeptide at R f = 0.62 andR f = 0.52, respectively. In the case of [14C]phenylalanine as tracer free phenylalanine was detected in addition to the tripeptide, which was released from GS1 by hydrolysis.To investigate the binding site of the tripeptide intermediate to gramicidin S synthetase 2, the GS2-d-Phe-Pro-Val-thioester complex was digested with CNBr and after fractionation of the CNBr peptides subsequently by S. aureus V8 protease. The active site peptide bearing the radiolabeled tripeptide was isolated in pure form by a three-step reversed phase HPLC purification protocol as described under “Experimental Procedures.” Because the thioester bond is unstable at neutral and alkaline pH, all cleavage and purification steps had to be performed in acidic medium (Fig. 2).Figure 2Purification of an active site peptide of GS2 bearing the tripeptide intermediate d-Phe-Pro-Val by reversed phase HPLC. The GS2-d-Phe-Pro-Val-tripeptide complex was digested chemically and enzymatically as indicated under “Experimental Procedures.” The labeled active site peptide was isolated in pure form by successive reversed phase HPLC steps. CNBr fragments were loaded onto a BioSil 304-10 column and separated with a linear gradient of acetonitrile from 0 to 70% in 150 min (A). The peptide fraction containing the labeled fragment was digested with endoproteinase GluC from S. aureus V8. The resulting fragments were loaded onto a BioSil 318-10 column and separated with a linear gradient of acetonitrile from 6 to 48% in 150 min (B). Fractions containing the radioactivity were rechromatographed on a μRPC SC 2.1/10 column using a Amersham Pharmacia Biotech Smart System and fractionated in the same way as under A (C).View Large Image Figure ViewerDownload (PPT)By liquid phase sequencing of the purified radiolabeled active site peptides containing eitherl-[14C]phenylalanine orl-[14C]valine as tracer we obtained the following result. 1LF2GP3G4H5ΔS6L7R8A9XSEQUENCE I The sequence shown in the upper row corresponds to the active site peptide of the thiolation center of GS2 for valine, which can be discriminated from the other thioester binding sites of gramicidin S synthetase by its arginine in position 7 instead of a lysine. This site was previously characterized by affinity labeling and analysis of the isolated peptide fragment (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar, 19Stein T. Vater J. Kruft V. Wittmann-Liebold B. Franke P. Panico Maria Mc Dowell R. Morris H.R. FEBS Lett. 1994; 340: 39-44Crossref PubMed Scopus (58) Google Scholar) with the exception that in position 5 a dehydroalanine was found instead of a serine as derived from the gene sequence (21Turgay K. Krause M. Marahiel M.A. Mol. Microbiol. 1992; 6: 529-546Crossref PubMed Scopus (171) Google Scholar). In previous experiments it has been demonstrated that this modification originates from an elimination reaction at the active site serine because of the alkaline conditions during Edman degradation (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar, 19Stein T. Vater J. Kruft V. Wittmann-Liebold B. Franke P. Panico Maria Mc Dowell R. Morris H.R. FEBS Lett. 1994; 340: 39-44Crossref PubMed Scopus (58) Google Scholar). Furthermore we would expect to find a methionine in position 9 of this peptide, which we could not detect. This is probably because of its conversion to the homoserine lactone during CNBr fragmentation.In the first two Edman degradation steps, in addition to L and G, F and P were detected, indicating that the FPV tripeptide was in fact attached to our active site peptide. Ifl-[14C]phenylalanine was used, the tracer quantitatively eluted in the first Edman degradation step. In the case of l-[14C]valine the radiolabel appeared in the third step. However, the degradation product was not identical with the phenylthiohydantoin derivative of valine. Presumably valine was linked to a 4′-PPan carrier via a thioester bond.To identify the mode of attachment of the phenylalanyl-prolyl-valine intermediate at the thiolation site of GS2 for l-valine, we investigated the isolated active site peptide by MALDI mass spectrometry (Fig. 3 A). In the linear mode we found an intensive signal atm/z = 1587.0 for the quasi-molecular ion [M+H]+ of this peptide containingl-[14C]valine as tracer. Its mass is consistent with the sum of the masses calculated (a) for the active site peptide comprising Leu-2037 to Met-2045 (m/z = 941.1) as derived from the gene sequence with a homoserine lactone instead of methionine in position 2045, (b) for a 4′-PPan cofactor (m/z = 340.3), and (c) for the tripeptide intermediate phenylalanyl-prolyl-valine (m/z = 352.4 withl-[14C]valine as the label). A second signal appeared at m/z = 876.4 that corresponds to the mass of a valine active site peptide of GS2 from which the 4′-PPan-tripeptide adduct was eliminated, converting the serine at position 2041 to a dehydroalanine.Figure 3Analysis of the active site peptide of GS2 bearing the tripeptide intermediate d-Phe-Pro-Val by matrix-assisted laser desorption ionization mass spectrometry. A, the post source decay spectrum of this species shows the quasi-molecular ion [M+H]+ atm/z = 1587.0 together with the fragment ions. Representative signals were found atm/z = 974.1 and 875.9, which could be attributed to the phosphorylated and the dephosphorylated, dehydrated active site peptide. B, structure of the dephosphorylated, dehydrated active site nonapeptide (M Δ) derived from the fragment signals that represent series of N- and C-terminal sequence ions. Masses of histidine-directed internal fragment ions indicated by an asterisk were also used for sequence determination.View Large Image Figure ViewerDownload (PPT)The structure of the active site peptide was determined by interpretation of the fragmentation data (Fig. 3) according to rules defined by Morris et al. (22Morris H.R. Panico M. Barber M. Bordoli R.S. Sedgwick R.D. Tyler A.N. Biochem. Biophys. Res. Commun. 1981; 101: 623-631Crossref PubMed Scopus (216) Google Scholar, 23Morris H.R. Panico M. Karplus A. Lloyd P.E. Riniker B. Nature. 1982; 300: 643-645Crossref PubMed Scopus (122) Google Scholar) and Biemann (24Biemann K. Methods Enzymol. 1990; 193: 455-479Crossref PubMed Scopus (326) Google Scholar). The fragmentation pattern of the parent ion atm/z = 1587.0 comprised a few series of N- and C-terminal (A, B, C, X, Y, and Z ions) as well as histidine-directed internal sequence ions (A*, B*, and C* ions), resulting in the amino acid sequence shown in Fig. 3 B. The dominant fragments are found at m/z ratios of 974.1 and 875.9. They correspond to the mass values of the phosphorylated and the dephosphorylated, dehydrated form of the active site peptide, respectively, with a dehydroalanine in position 5. The mass at m/z = 893.8 can be assigned to the dephosphorylated form of the active site peptide with serine in position 5. Furthermore the mass at m/z = 120.6 was attributed to the immonium ions of phenylalanine. The B ion of the dipeptide phenylalanyl-proline was detected at anm/z ratio of 245.8. The mass atm/z = 612.9 can be assigned to the dephosphorylated Pan-valyl-prolyl-phenylalanine adduct.We also analyzed the fragment pattern of the ion atm/z = 876.4. It is remarkably similar to that of the parent ion at m/z = 1587.0 and yields the same amino acid sequence (Fig. 3).The results obtained by amino acid analysis agree well with our sequence data. The active site peptide contained 1 mol each of β-alanine and taurine, the latter being an oxidation product of cysteamine corroborating the presence of an attached 4′-PPan substituent.In conclusion, evidence was obtained by sequence analysis, MALDI mass spectrometry, and amino acid analysis that the tripeptided-Phe-Pro-Val in the biosynthesis of gramicidin S is attached to the 4′-phosphopantetheine cofactor of the thiotemplate site for l-valine. In this way we identified the binding site of a peptide synthetase for one of its peptide intermediates for the first time. Our results are in full agreement with the prediction of the multiple carrier model that the growing peptide chain in the elongation process is attached to the thiotemplate site of its C-terminal amino acid component. This work represents a fundamental contribution to the understanding of the mechanism of nonribosomal peptide biosynthesis. Multifunctional peptide synthetases like gramicidin S synthetase from Bacillus brevis ATCC 9999 catalyze the biosynthesis of their peptide products by a thiotemplate mechanism (1Lipmann F. Acc. Chem. Res. 1973; 6: 361-367Crossref Scopus (99) Google Scholar, 2Laland S.G. Zimmer T.-L. Essays Biochem. 1973; 9: 31-57PubMed Google Scholar, 3Kurahashi K. Annu. Rev. Biochem. 1974; 43: 445-459Crossref PubMed Scopus (59) Google Scholar, 4Laland S.G. Frøyshov Ø. Gilhuus-Moe C. Zimmer T.-L. Nat. New Biol. 1972; 239: 43-44Crossref PubMed Scopus (30) Google Scholar, 5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar). They activate their amino acid substrates in a two-step process involving aminoacyl adenylate formation and subsequent thioesterification at specific thiol groups (thiotemplates). Such multienzymes show a modular organization (6Kleinkauf H. von Döhren H. Eur. J. Biochem. 1996; 236: 335-351Crossref PubMed Scopus (283) Google Scholar, 7Marahiel M.A. Stachelhaus T. Mootz H.D. Chem. Rev. 1997; 97: 2651-2673Crossref PubMed Scopus (898) Google Scholar, 8von Döhren H. Keller U. Vater J. Zocher R. Chem. Rev. 1997; 97: 2675-2705Crossref PubMed Scopus (202) Google Scholar, 9Vater J. Stein T. Vollenbroich D. Kruft V. Wittmann-Liebold B. Franke P. Liu L. Zuber P. J. Protein Chem. 1997; 16: 557-564Crossref PubMed Scopus (23) Google Scholar). They are composed of homologous biosynthetic units (modules) consisting of 1000–1500 amino acid residues. Their arrangement along the multifunctional polypeptide chain is usually colinear to the sequence of the amino acid components in the peptide product. Such modules can be subdivided into three main domains responsible (a) for amino acid recognition, binding, and the primary activation as aminoacyl adenylates, (b) for the thiolation of the amino acid substrates, and (c) for peptide elongation, in some cases in concert with amino acid epimerization. Recently we demonstrated by affinity labeling of gramicidin S synthetase at its thiolation centers and analysis of isolated active site peptides that each module of this multienzyme is equipped with its own 4′-phosphopantetheine (4′-PPan) 1The abbreviations used are: PPan, phosphopantetheine; GS1, phenylalanine racemase, EC 5.1.1.11(gramicidin S synthetase 1); GS2, gramicidin S synthetase 2; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization. 1The abbreviations used are: PPan, phosphopantetheine; GS1, phenylalanine racemase, EC 5.1.1.11(gramicidin S synthetase 1); GS2, gramicidin S synthetase 2; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization. cofactor (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar). The sulfhydryl group of the cofactors cysteamine component functions as the thiotemplate site for the specific amino acid substrate. Each of these mobile 4′-PPan carriers is connected in a phosphodiester linkage with a specific serine that is part of a strictly conserved consensus motif LGG(H/D)S(L/I). On the basis of these results we developed a multiple carrier model of nonribosomal peptide biosynthesis (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar). This new concept replaces the old version of the thiotemplate mechanism, which was proposed by several groups in the early seventies (1Lipmann F. Acc. Chem. Res. 1973; 6: 361-367Crossref Scopus (99) Google Scholar, 2Laland S.G. Zimmer T.-L. Essays Biochem. 1973; 9: 31-57PubMed Google Scholar, 3Kurahashi K. Annu. Rev. Biochem. 1974; 43: 445-459Crossref PubMed Scopus (59) Google Scholar, 4Laland S.G. Frøyshov Ø. Gilhuus-Moe C. Zimmer T.-L. Nat. New Biol. 1972; 239: 43-44Crossref PubMed Scopus (30) Google Scholar). The old model postulated a single central 4′-PPan carrier for peptide elongation that was assumed to interact during the elongation cycle with peripheral thiols charged with the amino acid substrates in thioester linkage. According to our new concept the growing peptide chain is assembled in a series of transpeptidation reactions by successive unidirectional interaction of the individual 4′-PPan carriers thioesterified with substrates or peptide intermediates. After transpeptidation the elongated peptide chain should be attached to the 4′-PPan cofactor specific for the binding of the C-terminal amino acid. This means that in the case of gramicidin S synthetase, for example, the di-, tri-, and tetrapeptide intermediate would be bound as thioesters to the 4′-PPan cofactor at the proline, valine, and ornithine reaction centers, respectively. Unfortunately, thed-Phe-Pro-dipeptide and thed-Phe-Pro-Val-Orn-tetrapeptide complexes of gramicidin S synthetase 2 (GS2) are unstable because of internal cyclization reactions (10von Döhren H. Kleinkauf H. von Döhren H. Peptide Antibiotics: Biosynthesis and Functions. de Gruyter, Berlin1982: 169-182Google Scholar, 11Vater J. Kleinkauf H. von Döhren H. Biochemistry of Peptide Antibiotics. de Gruyter, Berlin1990: 33-55Crossref Google Scholar, 12Vater J. Schlumbohm W. Palacz Z. Salnikow J. Gadow A. Kleinkauf H. Eur. J. Biochem. 1987; 163: 297-302Crossref PubMed Scopus (17) Google Scholar). On the other hand the GS2-d-Phe-Pro-Val-tripeptide intermediate shows an exceptionally high stability (13Gadow A. Vater J. Schlumbohm W. Palacz Z. Salnikow J. Kleinkauf H. Eur. J. Biochem. 1983; 132: 229-234Crossref PubMed Scopus (18) Google Scholar). Therefore, this species is optimally qualified to solve this question. In this communication we have identified the d-Phe-Pro-Val-attachment site of gramicidin S synthetase 2. This research is of general interest to understand the mechanism of nonribosomal peptide biosynthesis on the molecular level. RESULTS AND DISCUSSIONAs demonstrated in our previous studies each amino acid activating module of a peptide synthetase using the thiotemplate mechanism is equipped with its own 4′-PPan cofactor that functions as the thiolation site for the cognate amino acids substrate (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar, 19Stein T. Vater J. Kruft V. Wittmann-Liebold B. Franke P. Panico Maria Mc Dowell R. Morris H.R. FEBS Lett. 1994; 340: 39-44Crossref PubMed Scopus (58) Google Scholar). On the basis of these results a multiple carrier model was proposed that claims that the growing peptide chain is assembled during the elongation cycle in a series of transpeptidation steps. Each peptide intermediate should be bound to the 4′-PPan carrier of that module, which is responsible for the thiolation of the C-terminal amino acid component. To verify these predictions we investigated the binding site of GS2 for the tripeptided-phenylalanyl-prolyl-valine. This intermediate is well suited for such experiments, because of the high stability of its thioester bond with the corresponding reaction center of GS2 (13Gadow A. Vater J. Schlumbohm W. Palacz Z. Salnikow J. Kleinkauf H. Eur. J. Biochem. 1983; 132: 229-234Crossref PubMed Scopus (18) Google Scholar). For this purpose we labeled gramicidin S synthetase with the radioactive tripeptide as outlined under “Experimental Procedures” using eitherl-[14C]phenylalanine orl-[14C]valine as tracer.Tripeptide formation was monitored by TLC on silica gel DC 60 plates atR f = 0.72 with butanol:acetic acid:water (4:1:1) as the mobile phase (Fig. 1). Control samples contained the same reaction mixture from whichl-proline was omitted. Under these conditions phenylalanine and valine were incorporated into GS as thioesters. After cleavage with alkali, they were detected in free form on the TLC plates instead of the tripeptide at R f = 0.62 andR f = 0.52, respectively. In the case of [14C]phenylalanine as tracer free phenylalanine was detected in addition to the tripeptide, which was released from GS1 by hydrolysis.To investigate the binding site of the tripeptide intermediate to gramicidin S synthetase 2, the GS2-d-Phe-Pro-Val-thioester complex was digested with CNBr and after fractionation of the CNBr peptides subsequently by S. aureus V8 protease. The active site peptide bearing the radiolabeled tripeptide was isolated in pure form by a three-step reversed phase HPLC purification protocol as described under “Experimental Procedures.” Because the thioester bond is unstable at neutral and alkaline pH, all cleavage and purification steps had to be performed in acidic medium (Fig. 2).By liquid phase sequencing of the purified radiolabeled active site peptides containing eitherl-[14C]phenylalanine orl-[14C]valine as tracer we obtained the following result. 1LF2GP3G4H5ΔS6L7R8A9XSEQUENCE I The sequence shown in the upper row corresponds to the active site peptide of the thiolation center of GS2 for valine, which can be discriminated from the other thioester binding sites of gramicidin S synthetase by its arginine in position 7 instead of a lysine. This site was previously characterized by affinity labeling and analysis of the isolated peptide fragment (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar, 19Stein T. Vater J. Kruft V. Wittmann-Liebold B. Franke P. Panico Maria Mc Dowell R. Morris H.R. FEBS Lett. 1994; 340: 39-44Crossref PubMed Scopus (58) Google Scholar) with the exception that in position 5 a dehydroalanine was found instead of a serine as derived from the gene sequence (21Turgay K. Krause M. Marahiel M.A. Mol. Microbiol. 1992; 6: 529-546Crossref PubMed Scopus (171) Google Scholar). In previous experiments it has been demonstrated that this modification originates from an elimination reaction at the active site serine because of the alkaline conditions during Edman degradation (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar, 19Stein T. Vater J. Kruft V. Wittmann-Liebold B. Franke P. Panico Maria Mc Dowell R. Morris H.R. FEBS Lett. 1994; 340: 39-44Crossref PubMed Scopus (58) Google Scholar). Furthermore we would expect to find a methionine in position 9 of this peptide, which we could not detect. This is probably because of its conversion to the homoserine lactone during CNBr fragmentation.In the first two Edman degradation steps, in addition to L and G, F and P were detected, indicating that the FPV tripeptide was in fact attached to our active site peptide. Ifl-[14C]phenylalanine was used, the tracer quantitatively eluted in the first Edman degradation step. In the case of l-[14C]valine the radiolabel appeared in the third step. However, the degradation product was not identical with the phenylthiohydantoin derivative of valine. Presumably valine was linked to a 4′-PPan carrier via a thioester bond.To identify the mode of attachment of the phenylalanyl-prolyl-valine intermediate at the thiolation site of GS2 for l-valine, we investigated the isolated active site peptide by MALDI mass spectrometry (Fig. 3 A). In the linear mode we found an intensive signal atm/z = 1587.0 for the quasi-molecular ion [M+H]+ of this peptide containingl-[14C]valine as tracer. Its mass is consistent with the sum of the masses calculated (a) for the active site peptide comprising Leu-2037 to Met-2045 (m/z = 941.1) as derived from the gene sequence with a homoserine lactone instead of methionine in position 2045, (b) for a 4′-PPan cofactor (m/z = 340.3), and (c) for the tripeptide intermediate phenylalanyl-prolyl-valine (m/z = 352.4 withl-[14C]valine as the label). A second signal appeared at m/z = 876.4 that corresponds to the mass of a valine active site peptide of GS2 from which the 4′-PPan-tripeptide adduct was eliminated, converting the serine at position 2041 to a dehydroalanine.Figure 3Analysis of the active site peptide of GS2 bearing the tripeptide intermediate d-Phe-Pro-Val by matrix-assisted laser desorption ionization mass spectrometry. A, the post source decay spectrum of this species shows the quasi-molecular ion [M+H]+ atm/z = 1587.0 together with the fragment ions. Representative signals were found atm/z = 974.1 and 875.9, which could be attributed to the phosphorylated and the dephosphorylated, dehydrated active site peptide. B, structure of the dephosphorylated, dehydrated active site nonapeptide (M Δ) derived from the fragment signals that represent series of N- and C-terminal sequence ions. Masses of histidine-directed internal fragment ions indicated by an asterisk were also used for sequence determination.View Large Image Figure ViewerDownload (PPT)The structure of the active site peptide was determined by interpretation of the fragmentation data (Fig. 3) according to rules defined by Morris et al. (22Morris H.R. Panico M. Barber M. Bordoli R.S. Sedgwick R.D. Tyler A.N. Biochem. Biophys. Res. Commun. 1981; 101: 623-631Crossref PubMed Scopus (216) Google Scholar, 23Morris H.R. Panico M. Karplus A. Lloyd P.E. Riniker B. Nature. 1982; 300: 643-645Crossref PubMed Scopus (122) Google Scholar) and Biemann (24Biemann K. Methods Enzymol. 1990; 193: 455-479Crossref PubMed Scopus (326) Google Scholar). The fragmentation pattern of the parent ion atm/z = 1587.0 comprised a few series of N- and C-terminal (A, B, C, X, Y, and Z ions) as well as histidine-directed internal sequence ions (A*, B*, and C* ions), resulting in the amino acid sequence shown in Fig. 3 B. The dominant fragments are found at m/z ratios of 974.1 and 875.9. They correspond to the mass values of the phosphorylated and the dephosphorylated, dehydrated form of the active site peptide, respectively, with a dehydroalanine in position 5. The mass at m/z = 893.8 can be assigned to the dephosphorylated form of the active site peptide with serine in position 5. Furthermore the mass at m/z = 120.6 was attributed to the immonium ions of phenylalanine. The B ion of the dipeptide phenylalanyl-proline was detected at anm/z ratio of 245.8. The mass atm/z = 612.9 can be assigned to the dephosphorylated Pan-valyl-prolyl-phenylalanine adduct.We also analyzed the fragment pattern of the ion atm/z = 876.4. It is remarkably similar to that of the parent ion at m/z = 1587.0 and yields the same amino acid sequence (Fig. 3).The results obtained by amino acid analysis agree well with our sequence data. The active site peptide contained 1 mol each of β-alanine and taurine, the latter being an oxidation product of cysteamine corroborating the presence of an attached 4′-PPan substituent.In conclusion, evidence was obtained by sequence analysis, MALDI mass spectrometry, and amino acid analysis that the tripeptided-Phe-Pro-Val in the biosynthesis of gramicidin S is attached to the 4′-phosphopantetheine cofactor of the thiotemplate site for l-valine. In this way we identified the binding site of a peptide synthetase for one of its peptide intermediates for the first time. Our results are in full agreement with the prediction of the multiple carrier model that the growing peptide chain in the elongation process is attached to the thiotemplate site of its C-terminal amino acid component. This work represents a fundamental contribution to the understanding of the mechanism of nonribosomal peptide biosynthesis. As demonstrated in our previous studies each amino acid activating module of a peptide synthetase using the thiotemplate mechanism is equipped with its own 4′-PPan cofactor that functions as the thiolation site for the cognate amino acids substrate (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar, 19Stein T. Vater J. Kruft V. Wittmann-Liebold B. Franke P. Panico Maria Mc Dowell R. Morris H.R. FEBS Lett. 1994; 340: 39-44Crossref PubMed Scopus (58) Google Scholar). On the basis of these results a multiple carrier model was proposed that claims that the growing peptide chain is assembled during the elongation cycle in a series of transpeptidation steps. Each peptide intermediate should be bound to the 4′-PPan carrier of that module, which is responsible for the thiolation of the C-terminal amino acid component. To verify these predictions we investigated the binding site of GS2 for the tripeptided-phenylalanyl-prolyl-valine. This intermediate is well suited for such experiments, because of the high stability of its thioester bond with the corresponding reaction center of GS2 (13Gadow A. Vater J. Schlumbohm W. Palacz Z. Salnikow J. Kleinkauf H. Eur. J. Biochem. 1983; 132: 229-234Crossref PubMed Scopus (18) Google Scholar). For this purpose we labeled gramicidin S synthetase with the radioactive tripeptide as outlined under “Experimental Procedures” using eitherl-[14C]phenylalanine orl-[14C]valine as tracer. Tripeptide formation was monitored by TLC on silica gel DC 60 plates atR f = 0.72 with butanol:acetic acid:water (4:1:1) as the mobile phase (Fig. 1). Control samples contained the same reaction mixture from whichl-proline was omitted. Under these conditions phenylalanine and valine were incorporated into GS as thioesters. After cleavage with alkali, they were detected in free form on the TLC plates instead of the tripeptide at R f = 0.62 andR f = 0.52, respectively. In the case of [14C]phenylalanine as tracer free phenylalanine was detected in addition to the tripeptide, which was released from GS1 by hydrolysis. To investigate the binding site of the tripeptide intermediate to gramicidin S synthetase 2, the GS2-d-Phe-Pro-Val-thioester complex was digested with CNBr and after fractionation of the CNBr peptides subsequently by S. aureus V8 protease. The active site peptide bearing the radiolabeled tripeptide was isolated in pure form by a three-step reversed phase HPLC purification protocol as described under “Experimental Procedures.” Because the thioester bond is unstable at neutral and alkaline pH, all cleavage and purification steps had to be performed in acidic medium (Fig. 2). By liquid phase sequencing of the purified radiolabeled active site peptides containing eitherl-[14C]phenylalanine orl-[14C]valine as tracer we obtained the following result. 1LF2GP3G4H5ΔS6L7R8A9XSEQUENCE I The sequence shown in the upper row corresponds to the active site peptide of the thiolation center of GS2 for valine, which can be discriminated from the other thioester binding sites of gramicidin S synthetase by its arginine in position 7 instead of a lysine. This site was previously characterized by affinity labeling and analysis of the isolated peptide fragment (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar, 19Stein T. Vater J. Kruft V. Wittmann-Liebold B. Franke P. Panico Maria Mc Dowell R. Morris H.R. FEBS Lett. 1994; 340: 39-44Crossref PubMed Scopus (58) Google Scholar) with the exception that in position 5 a dehydroalanine was found instead of a serine as derived from the gene sequence (21Turgay K. Krause M. Marahiel M.A. Mol. Microbiol. 1992; 6: 529-546Crossref PubMed Scopus (171) Google Scholar). In previous experiments it has been demonstrated that this modification originates from an elimination reaction at the active site serine because of the alkaline conditions during Edman degradation (5Stein T. Vater J. Kruft V. Otto A. Wittmann-Liebold B. Franke P. Panico M. McDowell R. Morris H.R. J. Biol. Chem. 1996; 271: 15426-15435Google Scholar, 19Stein T. Vater J. Kruft V. Wittmann-Liebold B. Franke P. Panico Maria Mc Dowell R. Morris H.R. FEBS Lett. 1994; 340: 39-44Crossref PubMed Scopus (58) Google Scholar). Furthermore we would expect to find a methionine in position 9 of this peptide, which we could not detect. This is probably because of its conversion to the homoserine lactone during CNBr fragmentation. In the first two Edman degradation steps, in addition to L and G, F and P were detected, indicating that the FPV tripeptide was in fact attached to our active site peptide. Ifl-[14C]phenylalanine was used, the tracer quantitatively eluted in the first Edman degradation step. In the case of l-[14C]valine the radiolabel appeared in the third step. However, the degradation product was not identical with the phenylthiohydantoin derivative of valine. Presumably valine was linked to a 4′-PPan carrier via a thioester bond. To identify the mode of attachment of the phenylalanyl-prolyl-valine intermediate at the thiolation site of GS2 for l-valine, we investigated the isolated active site peptide by MALDI mass spectrometry (Fig. 3 A). In the linear mode we found an intensive signal atm/z = 1587.0 for the quasi-molecular ion [M+H]+ of this peptide containingl-[14C]valine as tracer. Its mass is consistent with the sum of the masses calculated (a) for the active site peptide comprising Leu-2037 to Met-2045 (m/z = 941.1) as derived from the gene sequence with a homoserine lactone instead of methionine in position 2045, (b) for a 4′-PPan cofactor (m/z = 340.3), and (c) for the tripeptide intermediate phenylalanyl-prolyl-valine (m/z = 352.4 withl-[14C]valine as the label). A second signal appeared at m/z = 876.4 that corresponds to the mass of a valine active site peptide of GS2 from which the 4′-PPan-tripeptide adduct was eliminated, converting the serine at position 2041 to a dehydroalanine. The structure of the active site peptide was determined by interpretation of the fragmentation data (Fig. 3) according to rules defined by Morris et al. (22Morris H.R. Panico M. Barber M. Bordoli R.S. Sedgwick R.D. Tyler A.N. Biochem. Biophys. Res. Commun. 1981; 101: 623-631Crossref PubMed Scopus (216) Google Scholar, 23Morris H.R. Panico M. Karplus A. Lloyd P.E. Riniker B. Nature. 1982; 300: 643-645Crossref PubMed Scopus (122) Google Scholar) and Biemann (24Biemann K. Methods Enzymol. 1990; 193: 455-479Crossref PubMed Scopus (326) Google Scholar). The fragmentation pattern of the parent ion atm/z = 1587.0 comprised a few series of N- and C-terminal (A, B, C, X, Y, and Z ions) as well as histidine-directed internal sequence ions (A*, B*, and C* ions), resulting in the amino acid sequence shown in Fig. 3 B. The dominant fragments are found at m/z ratios of 974.1 and 875.9. They correspond to the mass values of the phosphorylated and the dephosphorylated, dehydrated form of the active site peptide, respectively, with a dehydroalanine in position 5. The mass at m/z = 893.8 can be assigned to the dephosphorylated form of the active site peptide with serine in position 5. Furthermore the mass at m/z = 120.6 was attributed to the immonium ions of phenylalanine. The B ion of the dipeptide phenylalanyl-proline was detected at anm/z ratio of 245.8. The mass atm/z = 612.9 can be assigned to the dephosphorylated Pan-valyl-prolyl-phenylalanine adduct. We also analyzed the fragment pattern of the ion atm/z = 876.4. It is remarkably similar to that of the parent ion at m/z = 1587.0 and yields the same amino acid sequence (Fig. 3). The results obtained by amino acid analysis agree well with our sequence data. The active site peptide contained 1 mol each of β-alanine and taurine, the latter being an oxidation product of cysteamine corroborating the presence of an attached 4′-PPan substituent. In conclusion, evidence was obtained by sequence analysis, MALDI mass spectrometry, and amino acid analysis that the tripeptided-Phe-Pro-Val in the biosynthesis of gramicidin S is attached to the 4′-phosphopantetheine cofactor of the thiotemplate site for l-valine. In this way we identified the binding site of a peptide synthetase for one of its peptide intermediates for the first time. Our results are in full agreement with the prediction of the multiple carrier model that the growing peptide chain in the elongation process is attached to the thiotemplate site of its C-terminal amino acid component. This work represents a fundamental contribution to the understanding of the mechanism of nonribosomal peptide biosynthesis. We are indebted to Prof. R. M. Kamp, who made an amino acid analysis system available for our investigations. We thank G. Haeselbarth for peptide sequence analysis." @default.
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W2011719326 title "Characterization of the Binding Site of the Tripeptide Intermediated-Phenylalanyll-Prolyl-l-Valine in Gramicidin S Biosynthesis" @default.
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