Peptidyl Transfers in Gramicidin S Biosynthesis From Enzyme-Bound Thioester Intermediates

Gevers, Wieland; Kleinkauf, Horst; Lipmann, Fritz

doi:10.1073/pnas.63.4.1335

Cited by 133 publications

(83 citation statements)

References 11 publications

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“…Above pH 7 the rate of these reactions increases rapidly. This has also been confirmed for the reactive thioester intermediates involved in the biosynthesis of gramicidin S by Gevers et al [12]. In this work the cleavage of the thioester complexes of gramicidin S synthetase has been studied in neutral buffer solutions under mild conditions.…”

Section: Discussionsupporting

confidence: 71%

“…This procedure yields fully active synthetase and is, therefore, superior to the usually applied cleavage with alkali, performic acid or hydroxylamine [ 12,271 which results in complete deactivation of the multienzyme. The reaction centers of GS2 also remain catalytically competent, when the thiols at the surface of the synthetase are modified with SH inhibitors, like N-ethylmaleimide or iodoacetamide, following protection of the active thiol groups by substrate derivatization.…”

Section: Discussionmentioning

confidence: 95%

“…Aliquots of soluIn this study the stability of the reactive thioester-bound intermediates formed during the biosynthesis of gramicidin S was investigated. It is known that these complexes are stable in acidic medium and are efficiently hydrolyzed in alkaline solutions [12]. In Fig.…”

Section: Assay Proceduresmentioning

confidence: 99%

“…Formation of reactive thioesters of gramicidin S synthetase with substrate amino acids and peptide intermediates was accomplished essentially as described previously [12,13]. The concentration of the components in the reaction mixture were 5 -10 pM of a substrate amino acid, 2 mM ATP, 10 mM MgCI,, 1 mM EDTA and 1 mM dithioerythritol in 20 mM sodium phosphate buffer, pH 7.2.…”

Section: Assay Proceduresmentioning

confidence: 99%

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Gramicidin S Synthetase. Stability of Reactive Thioester Intermediates and Formation of 3-Amino-2-piperidone

Gadow¹,

Vater²,

Schlumbohm³

et al. 1983

Eur J Biochem

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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 recoverd. 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 "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.Specific features of the biosynthesis of certain classes of peptide antibiotics, as for example the gramicidins, tyrocidines or bacitracins, are the activation of substrate amino acids and (Tokyo, Japan). Column materials were AcA 34 from LKB and DEAE-cellulose DE 52 from Whatman.intermediate peptides on thiotemplates of multifunctional synthetases [l-61. Such multienzymes activate their substrate amino acids in a two-step mechanism involving aminoacyl adenylate and thioester formation. It is postulated [7] that the activated units are linked together in a series of transpeptidation and transthiolation reactions by interaction of an internal central phosphopantetheine carrier with the peripheral thiol groups at the reaction centers.At present only very limited knowledge is available on the architecture and spacial organization of these templates, as well as on the specific properties of the reactive thiols at the functional units of these multienzymes. In particular, information on the reactivity and stability of the intermediate thioester complexes is essential for labelling of the reaction centers followed by protein-chemical and physicochemical studies.The aim of this study, therefore, was (a) to investigate the hydrolytic stability of the reactive thioester complexes involved in the biosynthesis of gramicidin S, the cyclic decapeptide antibiotic [~Phe-Pro-Val-Om-Leu], from Bacillus brevis ATCC 9999, and (b) to develop reaction conditions for a specific modification of the peripheral thiol groups of gramicidin S synthetase. To a solution of 5 g (22.9 mmol) ornithine methyl ester dihydrochloride in 50 ml methanol, 6.28 ml (45.8 mmol) triethylamine were added and the mixture stirred for 3 h at 50 "C. The reaction mixture was evaporated to dryness in vacuo and the triethylammonium hydrochloride formed was removed by addition of chloroform. The residue was recrystallized from methanol by addition of a few drops of HCl/ether. Yield: 2.46 g (71.3 %); mp= 181 -182°C; [aid =16.2 (c=1.6; methanol...

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Section: Discussionsupporting

confidence: 71%

Section: Discussionmentioning

confidence: 95%

Section: Assay Proceduresmentioning

confidence: 99%

Section: Assay Proceduresmentioning

confidence: 99%

See 2 more Smart Citations

Gramicidin S Synthetase. Stability of Reactive Thioester Intermediates and Formation of 3-Amino-2-piperidone

Gadow¹,

Vater²,

Schlumbohm³

et al. 1983

Eur J Biochem

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“…bacitracin etc. (Gevers et al, 1969;Roskoski et al, pH 1970a,b;Fr0yshov, 1975), intermediate peptide (Table 1) inadenylates. the mycobacillin sequence, with enzyme fraction B…”

Section: Determination Ofprotein Concentrationmentioning

confidence: 99%

Functional characterization of constituent enzyme fractions of mycobacillin synthetase

Ghosh¹,

Majumder²,

Mukhopadhyay³

et al. 1985

Biochemical Journal

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The enzyme fraction A, a constituent enzyme of the three-fraction enzyme mycobacillin synthetase, independently and sequentially activated five amino acids starting from L-proline, producing the pentapeptide Pro(Asp1,Glu1,Tyr1)Asp. The fractions B and C were unable to function independently. However, the fraction B synthesized the nonapeptide Pro(Asp3,Glu1,Tyr2,Ser1)Leu, sequentially activating the pentapeptide and next four amino acids, whereas the fraction C synthesized mycobacillin by the sequential activation of the nonapeptide and the remaining four amino acids. The pH optima of the above enzymes are almost identical (pH 7.8), but their Km values are a little different.

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Structural consequences ofD-amino acids in collagen triple-helical peptides

Shah¹,

Brodsky²,

Kirkpatrick

et al. 1999

Biopolymers

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Peptidyl Transfers in Gramicidin S Biosynthesis From Enzyme-Bound Thioester Intermediates

Cited by 133 publications

References 11 publications

Gramicidin S Synthetase. Stability of Reactive Thioester Intermediates and Formation of 3-Amino-2-piperidone

Gramicidin S Synthetase. Stability of Reactive Thioester Intermediates and Formation of 3-Amino-2-piperidone

Functional characterization of constituent enzyme fractions of mycobacillin synthetase

Structural consequences ofD-amino acids in collagen triple-helical peptides

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