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Background: Bacillus subtilis strains produce a broad spectrum of lipopeptides that are potent biosurfactants and have specific antimicrobial and antiviral activities. The cyclic lipodecapeptide fengycin is one such compound. Although the fengycin biosynthetic genes in B. subtilis 168 (pps genes) and F29-3 (fen genes) have been well characterized, only limited information is available about the biochemical features of the fengycin synthetase multienzyme system., Results: Five multifunctional peptide synthetases (Fen1-5) that catalyze biosynthesis of the peptide portion of fengycin have been purified from crude extracts of the B. subtilis b213 and A1/3 strains. These enzymes activate all fengycin amino-acid components as aminoacyl adenylates or aminoacyl thioesters. Fen1, Fen2 and Fen3 are each approximately 286 kDa, Fen4 is approximately 400 kDa and Fen 5 is approximately 140kDa; each enzyme activates a different set of L-amino acids. A five-gene cluster (fen1-5) was detected in the B. subtilis A1/3 genome that shows high homology to the pps and fen genes in B. subtilis strains 168 and F29-3. Disruption of fen4 resulted in a loss of fengycin production. The fengycin synthetase enzymes isolated from B. subtilis b213 were assigned to the corresponding A1/3 fen genes by their amino-terminal sequences., Conclusions: The structural and functional organization of the fengycin synthetase system from B. subtilis b213 has been characterized in detail and correlated with the corresponding pps and fen genes in B. subtilis strains 168, A1/3 and F29-3. Biosynthesis of the peptide part of fengycin involves five multifunctional modular proteins that assemble the lipopeptide chain using a nonribosomal, multiple carrier thiotemplate mechanism.

The nearest neighbourhood of pigment-protein complexes within Photosystem II (PSII) membrane fragments has been studied by means of chemical cross-linking with o-phthalaldehyde (OPA) in conjunction with protein-chemical techniques. By means of OPA-induced cross-linking a major conjugate of about 60 kDa has been identified. This conjugate was shown to consist of two pigment-protein complexes of light-harvesting complex II (LHC II), Lhc b1 (CP27) and Lhc b4 (CP29) by means of SDS/PAGE in combination with an immunological analysis using mAbs directed against Lhc b4 and by matrix-assisted-laser-desorption-ionization mass spectrometry (MALDI-MS) and sequence analysis of peptides derived from a proteolytic digest of the conjugate. Domains of Lhc bl and Lhc b4 have been localized to a distance of not more than 5 A within LHC II. Our results are discussed in the light of recent models on the topography of the two subunits within the antenna system of Photosystem II.

Natural surfactin is a mixture of cyclic lipopeptides built from variants of a heptapeptide and a beta-hydroxy fatty acid with chain lengths of 13-15 carbon atoms. The lipopeptide biosurfactant was produced by Bacillus subtilis OKB 105 and part of the material subjected to esterification of its Glu and Asp residues. High-resolution preparative reversed phase HPLC on EnCaPharm 100 of surfactin and its monomethyl and dimethyl esters yielded 44 fractions which were characterized by NMR and MS methods. Among the separated isoforms are the known surfactin variants with l-Leu, l-Val, or l-Ile in position 7 of the peptide ring and three hitherto unknown variants showing replacements of the leucine residues in position 2 and/or 7 by l-Val and l-Ile. Our work makes available lipoheptapeptide compounds with modified structures and different hydrophobicities which promise to have potential for biotechnological and pharmaceutical applications. Copyright 1998 Academic Press.

The antiviral activity of surfactin, a cyclic lipopeptide antibiotic and biosurfactant produced by Bacillus subtilis, was determined for a broad spectrum of different viruses, Semliki Forest virus (SFV), herpes simplex virus (HSV-1, HSV-2), suid herpes virus (SHV-1), vesicular stomatitis virus (VSV), simian immunodeficiency virus (SIV), feline calicivirus (FCV), murine encephalomyocarditis virus (EMCV). In vitro experiments showed biphasic virus inactivation kinetics for enveloped viruses during treatment. Inactivation of enveloped viruses, especially herpes- and retroviruses, was much more efficient than that of non-enveloped viruses. For those viruses susceptible to its action, surfactin was active at 25 microM in medium containing 5% fetal calf serum (FCS). Concentrations up to 80 microM of surfactin led to a titre reduction of >4.4 log10 CCID50/ml for HSV-1 in 15 min and for SIV and VSV in 60 min. The inactivation rate increased linearly with the incubation temperature by a factor 2.4/10 degrees C and logarithmically with the concentration. Serum components, probably proteins and/or lipids, influence the effective surfactin concentration. A disruption of the viral lipid membrane and partially of the capsid was observed by electron microscopy. These findings suggest that the antiviral action, postulated also in other investigations, seems to be due to a physicochemical interaction of the membrane-active surfactant with the virus lipid membrane. Surfactin may be useful for application in virus safety enhancement of biotechnological and pharmaceutical products., (Copyright 1997 The International Association of Biological Standardization.)

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.

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].

The reactive serine of the TGGHSL thioester binding motif of the first amino acid-activating domain of surfactin synthetase was replaced by alanine using site-directed mutagenesis. The multienzyme from cells of the resulting mutant lost its ability for thioester formation with L-Glu and was therefore inactive in surfactin production. The thiolation reactions catalyzed by the other amino acid-activating domains of surfactin synthetase were not affected by the mutation. The results show that L-Glu is activated at the first domain of surfactin synthetase, and give further evidence that a serine residue is essential for substrate amino acid activation at the reaction centers of peptide synthetases.

The absorption losses for 1.06-µm laser radiation in Nd:YAG laser rods that are due to stable color centers are investigated. Stable color centers are created by a two-photon absorption process caused by theultraviolet part of the Xe flash-lamp spectrum. The reverse process of color center annihilation that is due to a one-photon bleaching process is observed. A model explaining these processes is proposed, and rate equations describing the creation and the annihilation of color centers are developed.

A 30-kDa chlorophyll-a/b-binding protein was purified from photosystem II membrane fragments using Ca(2+)-chelating Sepharose 6B chromatography. The protein binds approximately four chlorophyll a molecules, one chlorophyll b molecule and carotenoids. Its 77-K fluorescence-emission spectrum exhibits a maximum at 680 +/- 1 nm. The protein has a high tendency to form a dimer in the presence of Ca2+.Ca2+ binding affects the low-temperature fluorescence-emission maximum, leading to a decrease in its intensity and a blue shift of 1 nm. Similar spectral changes were obtained in the presence of Mg2+, possibly indicating a common binding domain for both cations. We interpret these observations as cation-induced conformational changes of the protein, which were reversible upon subsequent incubation in EDTA. Evidence is presented for the involvement of carboxyl groups in the coordination sphere of the bivalent cations. The possible structural and functional role of the protein is discussed.

The lipopeptide antibiotic surfactin is a potent extracellular biosurfactant produced by various Bacillus subtilis strains. Biosynthesis of surfactin was studied in a cell-free system prepared from B. subtilis ATCC 21332 and OKB 105, which is a transformant producing surfactin in high yield [Nakano, M. M., Marahiel, M. A., & Zuber, P. (1988) J. Bacteriol. 170, 5662-5668]. Cell material was disintegrated by treatment with lysozyme and French press. A cell-free extract was prepared by ammonium sulfate fractionation, which catalyzed the formation of surfactin at the expense of ATP. Lipopeptide biosynthesis required the L-amino acid components of surfactin and D-3-hydroxytetradecanoyl-coenzyme A thioester. D-Leucine which is present in surfactin was not utilized but inhibited the biosynthetic process. The structure of surfactin, synthesized enzymatically in vitro, was confirmed by chromatographic comparison with the authentic compound and by amino acid analyses. An enzyme fraction was prepared by gel permeation chromatography which catalyzed ATP/pyrophosphate exchange reactions dependent on the component amino acids of surfactin. This enzyme fraction was capable of binding substrate amino acids covalently, probably via thioester linkages. The formation of these intermediates was inhibited by various thiol blocking reagents and phenylmethanesulfonyl fluoride. De novo synthesis of the lipopeptide was not observed with this partially purified enzyme fraction most likely due to the lack of an acyltransferase activity required for linking the beta-hydroxy fatty acid to the peptide moiety.

A photosystem II core complex was purified with high yield from spinach by solubilization with beta-dodecylmaltoside. The complex consisted of polypeptides with molecular mass 47, 43, 34, 31, 9 and 4 kDa and some minor components, as detected by silver-staining of polyacrylamide gels. There was no indication for the chlorophyll-a/b-binding, light-harvesting complex polypeptides. The core complex revealed electron-transfer activity (1,5-diphenylcarbazide----2,6-dichloroindophenol) of about 30 mumol reduced 2,6-dichloroindophenol/mg chlorophyll/h. The structural integrity was analyzed by electron microscopy. The detergent-solubilized protein complex has the shape of a triangular disk with a maximum diameter of 13 nm and a maximum height of 6.8 nm. The shape of this core complex differs considerably from that of cyanobacterial photosystem II membrane fragments, which are elongated particles. The structural differences between both the complexes of higher plants and cyanobacteria are discussed with special emphasis on their association with the antenna apparatus in the photosynthetic membranes.

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.

The stimulation of the carboxylase reaction by effectors of ribulose 1,5-bisphosphate carboxylase/oxygenase displays higher sensitivity towards pyridoxal 5'-pyridoxal 5'-phosphate inhibition than the catalytical process itself. Pyridoxal 5'-phosphate binding to the enzyme is not affected by the modulators 6-phosphogluconate and fructose 1,6-bisphosphate at low concentrations at which these agents stimulate the carboxylation rate. At higher concentrations these sugar phosphates protect the enzyme against pyridoxal 5'-phosphate inhibition in a similar fashion like the substrate ribulose 1,5-bisphosphate. Such protection experiments in combination with spectrophotometrical studies of pyridoxal 5'-phosphate binding demonstrate two binding states of ribulose 1,5-bisphosphate at the reaction centers of the enzyme with different requirements for Mg2+. 6-Phosphogluconate functions as protector only in the presence of Mg2+. Our results imply a competition between pyridoxal 5'-phosphate and substrate or effector sugar phosphates at the reaction centers of the spinach carboxylase. It is proposed that the pyridoxal 5'-phosphate inhibition of the stimulatory activity of these effectors originates from a modification of the regulatory sites of the enzyme caused by pyridoxal 5'-phosphate binding to the catalytical sites.

The biosynthesis of the lipopeptide antibiotic surfactin was studied in whole cells of Bacillus subtilis ATCC 21332 which incorporate 14C-labeled precursor amino acids directly into the product. [14C]Acetate appeared in the fatty acid portion of surfactin and was also partially converted into leucine. An enzyme was isolated and partially purified from a cell-free extract of the bacillus which catalyzes ATP-Pi-exchange reactions which are mediated by the amino acid components of surfactin. This activation pattern is consistent with a peptide synthesizing multienzyme which activates its substrate amino acids simultaneously as reactive aminoacyl phosphates.

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.

The amino-acid sequence of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Zea mays has been determined by alignment of peptides generated by digestion with trypsin, chymotrypsin, staphylococcal protease and thermolysin. The protein-chemically determined structure is in complete agreement with the nucleotide-derived sequence as published recently (Matsuoka et al. (1987) J. Biochem. 102, 673-676), but in addition possesses, however, sixteen experimentally verified dimorphies at various positions and possibly nine more for which evidence is tentatively pointing to two (or more) different expressed nuclear genes for the small subunit. These protein dimorphies represent a protein family corresponding to a gene family, the components of which have not been separated. The closest homologous polypeptide is the small subunit from wheat.

The cytochrome b6f complex of spinach chloroplasts was prepared with minor modification according to the method of E. Hurt and G. Hauska (1981) Eur. J. Biochem. 117, 591-599) replacing, however, the final ultracentrifugation step by hydroxyapatite chromatography as suggested by M. F. Doyle and C.-A Yu (1985) Biochem. Biophys. Res. Commun. 131, 700-706). The purified complex was partially dissociated by treatment with 4 M urea or 0.1% sodium dodecyl sulfate (SDS) in the absence of reducing agents. A binary subcomplex consisting of cytochrome f and the Rieske iron-sulfur protein was observed under these conditions by three different methods: (a) hydroxyapatite chromatography; (b) extraction with an isopropanol/water/trifluoroacetic acid mixture; and (c) gel filtration in the presence of low SDS concentrations. The subcomplex dissociated into its components by treatment with mercaptoethanol. These results suggest a close interaction of the cytochrome f with the Rieske protein involving SH groups which under reducing conditions leads to complete dissociation of the subcomplex.

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.