Structure of the cell walls of Micrococcus lysodeikticus.  III.  Isolation of a new peptide dimer, Nα-[L-alanyl-γ-(α-D-glutamylglycine)]-L-lysyl-D-alanine

Ghuysen, J. M.; Bricas, E.; Lache, Marvin; Leyh‐Bouille, Mélina

doi:10.1021/bi00844a030

Cited by 100 publications

(74 citation statements)

References 25 publications

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“…Free D-alanine was estimated by reaction with fluorodinitrobenzene (14,15). RESULTS Transpeptidase Activity of the R61 and R39 Enzymes at High Acceptor Concentrations.…”

Section: Methodsmentioning

confidence: 99%

Transpeptidase Activity of Streptomyces D-Alanyl-D Carboxypeptidases

Pollock

Ghuysen

Linder

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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In the presence of Na,NE-diacetyl-L-Lys- Paper electrophoresis was performed in 0.2 N acetic acid (pH 2.9), in acetic acid-l)yridine-water 0.33:4: 1000, pH 5.6, and in acetic acid-collidine-water 2.65:9.10: 1000, pH 7.Radioactive chromatograms or electrophoretograms were quantitatively analyzed by cutting strips (3.6 cm in width) into sections of 5-10 mm (12). The paper sections were placed in counting vials, 0.5 ml of water was added, and the vials were allowed to stand overnight. Bray's (13) dioxanescintillant (11 ml) was added and the samples were counted. In some experiments, samples were eluted before addition of the scintillant.Free D-alanine was estimated by reaction with fluorodinitrobenzene (14,15). RESULTSTranspeptidase Activity of the R61 and R39 Enzymes at High Acceptor Concentrations. Fig. la shows the rate of release of the C-terminal D-alanine from N',Nf-diacetyl-iLyS-D-Ala-D-Ala by R61 and R39 DD carboxypeptidases. Incubations were at 37°C in 17 mM phosphate buffer, pH 8, for the R61 enzyme and either in the same phosphate buffer or in 34 mi\M veronal buffer, pH 8.5, for the R39 enzyme; the final enzyme concentrations were 27 units/Al and 4 units/,l, respectively.The concentration of tripeptide was 1.5 mM; maximal hydrolysis was reached after about 90 min. Fig. 1 With both the R61 and R39 enzymes, -alanine and glycine were found to be acceptors, while iLalanine, even at a ratio to tripeptide of 100:1, was not an acceptor. At the 100:1 ratio of D-alanine or glycine to tripeptide, formation of either N',-N'-diacetyl-iLys-D-Ala-['4C]D-Ala or Na,NE-diacetyl-iLysn-Ala-[14C]Gly closely paralleled hydrolysis of the donor tripeptide in the absence of amino acid acceptor (Fig. lb and c, compare with a). After 90 min, at which time hydrolysis was 662 § To whom correspondence should be addressed.

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“…Free D-alanine was estimated by reaction with fluorodinitrobenzene (14,15). RESULTS Transpeptidase Activity of the R61 and R39 Enzymes at High Acceptor Concentrations.…”

Section: Methodsmentioning

confidence: 99%

Transpeptidase Activity of Streptomyces D-Alanyl-D Carboxypeptidases

Pollock

Ghuysen

Linder

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

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“…Hydrazinolysis and quantitative estimation of C-terminal amino acids were performed according to Ghuysen et at. [5] (see Materials and Methods). Leucine was found to be the principal C-terminal amino acid (86%) with a much smaller amount of valine (140/0), in accordance with the presence of a heptapeptide chain containing a C-terminal valine residue.…”

Section: Positiori Qf the Valinr Residuementioning

confidence: 99%

Isolation and characterization of a new variant of surfactin, the [Val7]surfactin

Peypoux

Bonmatin

Labbé

et al. 1991

European Journal of Biochemistry

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Reinvestigation of surfactin, a previously studied peptidolipid surfactant from Bacillus subtilis, by fast-atombombardment mass spectrometry and 'H-NMR spectroscopy, as well as by chemical methods, revealed the presence of a closely related second constituent. This new compound, [Val7]surfactin, differs from the known surfactin by the C-terminal amino acid residue which is valine instead of leucine.Surfactin is a peptidolipid produced by several strains of Bacillus subtilis which possesses a high surfactant activity [l]. The structure of surfactin has been determined as a macrolide containing seven a-amino acids and a homologous series of hydroxy fatty acids with 13,14 or 15 carbon atoms [2] (Fig. I).While studying strain S 499 of B. suhtilis, which produces an antifungal peptidolipid iturin A, we found that this strain gave a high yield of both iturin A and surfactin [3]. In the course of our studies on surfactin production with various substrates in the culture medium, the characterization of surfactin was carried out by chemical analysis and mass spectrometry. The mass spectrum of surfactins obtained from Landy medium [4] and from medium containing valine instead of glutamic acid showed some differences in molecular ion peaks suggesting a variation in the lipid or in the peptide moiety.The present paper characterizes a new variant of surfactin with a peptide sequence different from the known surfactin. MATERIALS AND METHODS Culture conditions and surfactin purificationSurfactin was produced by B. subtilis S 499 supplied by Dr L. Delcambe (CNPEM, Liige, Belgium). The seeded flasks, containing brain/heart infusion (bioMkrieux), were shaken for two days at 32°C. A 30-ml portion was used to inoculate the production medium (300ml in 2-1 Erlenmeyer flasks). The cultures were grown in the medium of Landy et al. [4] in a rotary shaker for five days at 32 "C. In order to obtain radioactive surfactin, 37 kBq sodium [l-'4C]acetate/ml medium were added at the middle of the exponential growth phase. At the end of growth, the culture was adjusted to pH 2.0 with 6 M HCl. After centrifugation, the pellets were neutralized and lyophilised. The peptidolipids were successively extracted with chloroforni/methanol(2: 1, by vol.) and with methanol.Surfactin was purified from the crude extract by two successive column chromatographies on silicic acid Bio-Sil HA 325 pm mesh (Bio-Rad). In the first chromatography, the elution was performed with chloroform/methanol/water (65 : 25: 4, by vol.), the elution volume of surfactin was 120 ml with a column (32 cm x 1.6 cm) containing 50 g silicic acid.The fractions containing surfactin were purified by a second chromatography on a column (27 cm x 1.6 cm) of silicic acid (40 g), eluting with a discontinuous gradient of methanol (0.7 -4.2%) in hexane/chloroform (36 : 64, by vol); the elution volume of surfactin was 260 ml. The fractions were analyzed by TLC on silica gel 60 (Merck) in chloroform/methanol/ water (65: 25:4, by vol.). Surfactin was detected as white spots by spraying with water a...

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“…L'aminoacide C-terminal est determine apres hydrazinolyse [20] sous forme de dinitrophenyl-derive par chromatographie sur couches minces de gel de d i c e 60 avec le solvant chloroforme/methanol/acide acetique (92/5/3, en vol. ).…”

Section: Sdquence Peptidiqueunclassified

Structure de la bacillomycine L, antibiotique de Bacillus subtils

Besson

Peypoux

Michel

et al. 1977

European Journal of Biochemistry

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The Structure of Bacillomycin L, an Antibiotic from Bacillus subtilisThe structure of bacillomycin L, an antifungal agent isolated from the culture medium of a strain of Bacillus subtilis, has been investigated. The peptide moiety contains one mole each of D-aspartic acid, L-aspartic acid, L-glutamine, L-serine, D-Serine, L-threonine, and D-tyrosine. The lipid moiety is a mixture of 3-amino-12-methyltridecanoic acid (46 %), 3-amino-12-methyltetradecanoic acid (38 %), 3-amino-14-methy1pentadecanoic acid (1 1 %), and two minor homologues.The peptide sequence and the cyclic structure were determined by structural analysis of the peptides obtained by mild acid hydrolysis.The molecular weight was determined by the thermoosmotic method ; this showed that bacillomycin L has a monomeric structure which is given in Formula 1.

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Structure of the cell walls of Micrococcus lysodeikticus. III. Isolation of a new peptide dimer, Nα-[L-alanyl-γ-(α-D-glutamylglycine)]-L-lysyl-D-alanine

Cited by 100 publications

References 25 publications

Transpeptidase Activity of Streptomyces D-Alanyl-D Carboxypeptidases

Transpeptidase Activity of Streptomyces D-Alanyl-D Carboxypeptidases

Isolation and characterization of a new variant of surfactin, the [Val7]surfactin

Structure de la bacillomycine L, antibiotique de Bacillus subtils

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