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The tripeptide intermediate D-Phe-Pro-Val in the biosynthesis of gramicidin S was labeled by incorporation of either L-[14C]phenylalanine or L-[14C]valine in an in vitro biosynthetic 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.

Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.

Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4'-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.

The biosynthesis of the decapeptide antibiotic gramicidin S in Bacillus brevis ATCC 9999 is catalyzed by a multienzyme system consisting of two multifunctional proteins, gramicidin S synthetase 1 and 2, encoded by the grsA and grsB genes, respectively. Gramicidin S synthetase 1 (phenylalanine racemase, EC 5.1.1.11, GS1) racemizes phenylalanine in the thioester-bound stage. The amount of 4'-phosphopantetheine liberated from highly purified GS1 was determined microbiologically using Lacto-bacillus plantarum as the test organism. It matches exactly with the amount of L-[14C]phenylalanine covalently incorporated by GS1 as thioester. The reaction center of GS1 for L-phenylalanine thiolation and racemization was labeled with [3H]iodoacetic acid. After tryptic fragmentation of the 3H-carboxymethylated enzyme, the active site peptide for thioester binding and racemization of phenylalanine was isolated in pure form by multistep methodology and investigated by sequence, amino acid, and mass spectrometric analysis. A 4'-phosphopantetheine carrier was found to be attached to the active site serine of the consensus motif LGGDSI forming the thiolation site of phenylalanine. These specific properties establish GS1 as a prototype of amino acid racemases using 4'-phosphopantetheine as a cofactor and yield further evidence that multiple Pan carriers are involved in gramicidin S formation. Our results are strong evidence for the "multiple carrier model" as a new concept of nonribosomal peptide biosynthesis at protein templates as recently proposed [Stein, T., et al. (1994) FEBS Lett. 340, 39-44].

Biosynthesis of gramicidinS in Bacillus brevis is catalysed by a multienzyme system consisting of two multifunctional proteins, gramicidinS synthetase 1 and 2 codified by the grsA and grsB genes, respectively. GramicidinS synthetase 2 shows a modular architecture of four amino acid-activating domains each containing a thioester binding motif LGG H/D S L/I highly conserved in its C-terminal region, as demonstrated by sequence analysis of the grsB gene [W. Schlumbohm et al. (1991) J. Biol. Chem. 266, 23135-23141]. This multienzyme was specifically labeled at the thioester binding site of L-valine with [3H]N-ethylmaleimide using a substrate protection technique. After enzymatic digestion a labeled active site peptide was isolated in pure form by multistep methodology. This fragment was identified by gas-phase sequencing as the active site peptide of the thiotemplate site for L-Val by comparison with the grsB gene sequence. By mass spectrometry in combination with amino acid analysis it was demonstrated that a 4'-phosphopantetheine carrier was attached to the active serine in this motif. Our results give evidence that multiple peripheral 4'-phosphopantetheine carriers are involved in the formation of gramicidinS in contrast to a central carrier arm as assumed in the original version of the thiotemplate mechanism. A 'Multiple Carrier Model' of nonribosomal peptide biosynthesis is proposed.

The condensing peptide forming multienzyme of gramicidin S synthetase (gramicidin S synthetase 2) was specifically labeled at its putative thiotemplate sites for L-valine and L-leucine by covalent incorporation of the 14C-labeled substrate amino acids. The thioester complexes of the multienzyme were digested with CNBr, Staphylococcus aureus V8 protease, and pepsin. Reaction center peptides containing the [14C]valine and [14C]leucine labels were isolated in pure form. They show a high degree of sequence similarity and contain the same consensus sequence LGGH/DXL. The labels were eliminated in the first Edman degradation step. A dehydroalanine was identified which can originate from either a cysteine or a serine. The comparison of the chemical results with the deduced amino acid sequence of the grsB gene encoding the gramicidin S synthetase 2 revealed that 4 such motifs are located within the gene structure, each of them being localized in the 3'-terminal region of one of 4 gene segments grsB1-B4. They have a size of approximately 2 kilobases and presumably code for the 4 amino acid activating domains of the synthetase. Surprisingly a serine was found at each putative substrate amino acid-binding position instead of a cysteine as postulated by the thiotemplate mechanism. Therefore the data suggest that active serine residues are involved in nonribosomal peptide syntheses of microbial peptides.

An investigation was made of the intermolecular forces which determine substrate recognition and binding as well as of the topography and localized environment of the different binding sites of the substrate amino acids of gramicidin S-synthetase (GSS) using substrate derivatives as molecular probes. It is demonstrated that among the aminoacyl adenylate binding sites of the heavy component of GSS the activation site of L-ornithine is distinguished by a relatively high substrate variability. The active centres of GSS are less restrictive for the activation of substrate analogues modified at the carboxyl group than for derivatives substituted at the alpha-amino group.

The reactive thioester complexes of gramicidin S synthetase with substrate amino acids and intermediate peptides are slowly hydrolyzed in neutral buffer solutions under mild conditions. Fully active enzyme is recovered. These processes are strongly accelerated by certain thiol protective agents. In the presence of 1 mM dithioerythritol the half-life times of these hydrolysis reactions are in the range of 1-90 h at 3 degrees C. The thioester complex of gramicidin S synthetase 2 (GS2, the heavy enzyme) with the tripeptide DPhe-Pro-Val is distinguished by the highest stability of all these intermediates. A different decomposition pattern is observed for the thioester complex of GS2 with LOrn. Here 3-amino-2-piperidone (cyclo-LOrn) is formed in a rapid cyclization reaction. This product specifically blocks the activation center of GS2 for LOrn at the thioester binding site. All other activation reactions of gramicidin S synthetase are unaffected. A procedure for a specific labelling of the reaction centers of the multienzyme is outlined.

In the biosynthesis of the cyclic decapeptide antibiotic gramicidin S, the constituent amino acids are activated by a two-step mechanism involving aminoacyl adenylate and thio ester formation which are both reversible processes. The dissociation constants (KD) for the gramicidin S synthetase-substrate amino acid-thio ester complexes are 100-1000-fold lower compared to the KM data of the preceding aminoacyl adenylate reactions. The affinity for these substrates is appreciably higher at the thio template sites than at the aminoacyl adenylate reaction centers. Therefore, the activation equilibria are quantitatively shifted toward thio ester formation. A set of thermodynamic parameters for the activation processes was determined from the temperature dependence of the KM and KD data. Reaction enthalpies were obtained from a van't Hoff analysis of these constants. delta G degree for the substrate activation reactions of the heavy enzyme of gramicidin S synthetase (GS 2) is predominantly controlled by entropy contributions. In contrast, the overall activation and concomitant racemization of phenylalanine by phenylalanine racemase (GS 1) are exothermic processes which are distinguished by a small negative reaction entropy.

The preparation of both enzymes of gramicidin S synthetase was efficiently improved by introduction of the fast protein liquid chromatography technique. High-resolution anion-exchange chromatography on Pharmacia Mono Q HR 5/5 was used as the final purification step. D-Phe-Pro-Val-cyclo-Orn was obtained as a product of the multienzyme by omission of L-leucine from the complete bioassay mixture. This tetrapeptide was formed by cyclization of the C-terminal ornithine to 3-amino-2-piperidone. It was identified and characterized by chromatographic and spectroscopic procedures using chemically synthesized reference compounds.

1. Chromatography on hydroxyapatite and on aminohexyl-Sepharose as well as isoelectric focusing were introduced as new effective purification procedures for phenylalanine racemase (EC 5.1.1.11). The enzyme preparations obtained were essentially homogeneous, as demonstrated by specific activity measurements and polyacrylamide gel electrophoresis. 2. The enzyme is not dissociable by sodium dodecyl sulfate. 3. Phenylalanine racemase is an acidic protein with an isoelectric point of approx. 4.6 (isoelectric focusing). 4. The Michaelis constants of L-Phe and D-Phe in the aminoacyl adenylate activation are 0.06 and 0.13 mM, respectively. 5. From our studies with structural analogues of phenylalanine we infer that the amino group of this amino acid is essential for its binding to the aminoacyl adenylate reaction center. The carboxyl group is not at all or only weakly bound. The benzene ring of phenylalanine which determines substrate recognition also seems to be of minor importance for substrate binding.

The amino-acid compositions of both enzymes of gramicidin S synthetase were determined. These proteins contain a high number of acidic amino-acid residues. Phenylalanine racemase, the light enzyme, was sequenced from the N-terminus until position 10. The kinetics of the thioester formation reactions were studied. The half-life times of these processes under substrate saturation conditions were found in the range between seconds and a few minutes. The valine activation at the heavy enzyme was detected as one of the rate-limiting steps of the biosynthesis of gramicidin S.

Six accessible sulfhydryl groups of the light component of gramicidin S synthetase (GS 1) were titrated with N-ethylmaleimide (Ma1NEt) as well as 3,3'-dithiobis(6-nitrobenzoic acid) (Nbs2). Twenty-four thiols were detected in the heavy enzyme (GS 2) using Ma1NEt as the modifier. Substrate amino acid-induced protection of GS 2 against deactivation by Ma1NEt indicates that in addition to the specific thiols at the thiotemplates of gramicidin S synthetase reactive SH groups are also involved in the primary aminoacyl adenylate activation reactions. Obviously 2 sulfhydryl groups are essential for the adenylation of each L-Pro, L-Val and L-Leu, while 3 thiols were detected for the ornithine activation. Agents like Ma1NEt or Nbs2 inhibit the thiolation of the substrate amino acids of gramicidin S synthetase and gramicidin S formation more severely than the aminoacyl adenylate activation reactions which are affected at 10-100-fold higher inhibitor concentrations. These processes can be studied separately if the thioester formation sites are inhibited at low concentrations of sulfhydryl inhibitors.

Dipeptides L-phenylalanyl-proline, D-phenylalanyl-proline, prolyl-valine, valyl-lysine, lysyl-leucine and leucyl-phenylalanine, derived from the sequence of gramicidin S, are substrates of the gramicidin S synthetase. When any of these dipeptides are used to replace the two corresponding amino acids in the reaction assay, cyclodecapeptide antibiotic synthesis occurs, and requires the whole multienzyme system. Active esters, like the thiophenyl and p-nitrophenyl esters of D-phenylalanyl-proline are unable to promote gramicidin S biosynthesis with the gramicidin S synthetase system or with the heavy enzyme alone.