Cell-Free Synthesis of Gramicidin S<sup>*</sup>

Tomino, Shiro; Yamada, Masao; Itoh, Hajime; Kurahashi, Kiyoshi

doi:10.1021/bi00860a037

Cited by 79 publications

(25 citation statements)

References 23 publications

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“…This can be concluded from the stability of aminoacyl thioesters [7, 601 and indeed, in studies on the formation of product from a labelled precursor, an initial lag phase has been found [61]. Some evidence for an inhibition of the enzyme upon mis-acylation has come from substrate protection studies performed to aid isolation of the active multienzyme [62].…”

Section: The Gramicidin S Cyclementioning

confidence: 99%

Nonribosomal biosynthesis of peptide antibiotics

Kleinkauf¹,

Döhren²

1990

European Journal of Biochemistry

311

161

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Peptide antibiotics are known to contain non-protein amino acids, D-amino acids, hydroxy acids, and other unusual constituents. In addition they may be modified by N-methylation and cyclization reactions. Their biosynthetic origin has been connected in many cases to an enzymatic system referred to as the 'thiotemplate multienzymic mechanism'. This mechanism includes the activation of the constituent residues as adenylates on the enzymic template, the acylation of specific template thiol groups, epimerization or N-methylation at this thioester stage, and polymerization in the sequence directed by the multienzymic structure with the aid of 4 -phosphopantetheine as a cofactor, including possible cyclization or terminal modification reactions. The reaction sequences leading to gramicidin S, tyrocidine. cyclosporine, bacitracin, polymyxin, actinomycin, enniatin, beauvericin, &(L-a-aminoadipyl)-L-cysteinyl-D-vahne and linear gramicidin are discussed. The structures of the multienzymes,' their genetic organization, the biological functions of these peptides and results on related systems are discussed.Although the enzymatic formation of essential peptides such as glutathione and pantetheine was already known in the 'preribosomal era', the elucidation of the biosynthesis of more complex peptides followed the unravelling of the genetic code in the sixties [l -31. A prediction of a poly-or multienzymatic pathway to peptides had been presented by Fritz Lipmann as early as 1954 [4] and a similar biosynthetic scheme of multienzymes as templates for polypeptides has now been verified for various types of peptides. Some of these studies have been reviewed elsewhere [5 -121, but recent results have advanced the field considerably. We therefore attempt to present here a more general view of this biosynthetic mechanism, and discuss the current areas of research.

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Section: The Gramicidin S Cyclementioning

confidence: 99%

Nonribosomal biosynthesis of peptide antibiotics

Kleinkauf¹,

Döhren²

1990

European Journal of Biochemistry

311

161

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“…In B. brevis, gramicidin S, an antibiotic decapeptide (D-Phe-L-Pro-L-Val-L-Orn-L-Leu)2, is formed by gramicidin S synthetase 1 (GS 1) (phenylalanine racemase) [EC 5.1.1.11] and gramicidin S synthetase 2 (GS 2) (L-Pro, L-Val, L-Orn, and L-Leu activating enzyme complex) (1,2). The biosynthesis of phenylalanine, valine, and leucine by aminotransferases may be essential for the formation of gramicidin S as well as the cell growth.…”

mentioning

confidence: 99%

Purification and Properties of Branched Chain Amino Acid Aminotransferase from Gramicidin S-Producing Bacillus brevis.

Kanda

Hori

Kurotsu

et al. 1995

Journal of Nutritional Science and Vitaminology, J Nutr Sci Vit

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SummaryThe branched chain amino acid aminotransferase [EC 2.6.1.42] was purified to a homogeneous state from a gramicidin S producing strain of Bacillus brevis. The enzyme had a molecular weight of about 93,000 and consisted of two identical subunits, each with a molecular weight of about 47,000. One pyridoxal phosphate is bound per subunit. In addition to branched chain amino acids, the enzyme uses L-phenylalanine and L-tryptophan as the amino donor, indicating that B. brevis branched chain amino acid aminotransferase has a broad substrate specificity for the amino donor. The enzyme utilized 2-oxoglutarate as the amino acceptor. The purified enzyme exhibits its absorption maxima at 332 and 427nm at neutral pH.

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“…Various microorganisms have now been found to produce antibiotic peptides nonribosomally (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Much of our present knowledge and our formulation (1) of nonribosomal peptide synthesis has been obtained from the study of the biosynthetic mechanisms of gramicidin S and tyrocidine, which are decapeptide antibiotics produced by the Bacillus brevis strains ATCC 9999 and ATCC 8185, respectively.…”

mentioning

confidence: 99%

“…The biosynthesis of gramicidin S requires two complementary enzymes, that of tyrocidine, three. Of these complementary enzymes, one for gramicidin S and two for tyrocidine biosynthesis are multienzymes containing pantetheine (7)(8)(9)(10)(11)(12)(13)15). The peptide synthesis proceeds with the ATP-driven activation of amino acids followed by attachment of the activated amino acids to the peripheral enzymic thiol groups; then, with the aid of enzymelinked pantetheine, condensation of the thioesterified amino acids ensues (1,(7)(8)(9).…”

mentioning

confidence: 99%

Isolation of amino acid activating subunit-pantetheine protein complexes: Their role in chain elongation in tyrocidine synthesis

Lee¹,

Lipmann²

1977

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

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Dissociation of the multienzymes of tyrocidine synthesis by prolonged incubation of crude extracts of Bacillus brevis (Dubos strain, ATCC 8185) has yielded, on Sephadex G-100 chromatography, two fractions of amino acid activating subunits, a larger one of 70,000 daltons and a smaller one of 90,000 dalto s; the latter was a complex consisting of the 70,000 dalton subunit and the pantetheine-carrying protein of about 20,000 daltons. When it dissociated, the intermediate enzyme, which activates three amino acids, contained two-thirds of the subunits in the 70,000 dalton and one-third in the 90,000 dalton fraction; the heavy enzyme, which activates six amino acids, contained five-sixths of the subunits in the former fraction and one-sixth in the latter. Both fractions showed ATP-PP1 exchange with all amino acids that are activated by the respective polyenzymes. With proline as an example, the 70,000 dalton subunit exhibited a single low-affinity binding site, which should correspond to the peripheral thiol acceptor site, whereas the 90,000 dalton subunit showed both a low-affinity binding site and an additional high-affinity site for proline; the high-affinity site is attributed to the pantetheine present on the pantetheine-carrying protein, and suggests that amino acids are translocated from the peripheral SH to the pantetheine-carrying moiety during chain elongation. This was confirmed by the observation that the 90,000 dalton complex, when incubated with the light enzyme in the presence of phenylalanine and proline, produced DPhe-Pro dipeptide that cyclized into DPhe-Pro diketopiperazine, but the 70,000 dalton activating subunit, when similarly incubated, did not. After subunit dissociation, however, no further elongation occurred after the transfer from phenylalanine to proline.Various microorganisms have now been found to produce antibiotic peptides nonribosomally (1-18). Much of our present knowledge and our formulation (1) of nonribosomal peptide synthesis has been obtained from the study of the biosynthetic mechanisms of gramicidin S and tyrocidine, which are decapeptide antibiotics produced by the Bacillus brevis strains ATCC 9999 and ATCC 8185, respectively. The biosynthesis of gramicidin S requires two complementary enzymes, that of tyrocidine, three. Of these complementary enzymes, one for gramicidin S and two for tyrocidine biosynthesis are multienzymes containing pantetheine (7-13, 15). The peptide synthesis proceeds with the ATP-driven activation of amino acids followed by attachment of the activated amino acids to the peripheral enzymic thiol groups; then, with the aid of enzymelinked pantetheine, condensation of the thioesterified amino acids ensues (1,(7)(8)(9). Both the involvement of two or more enzymes, including the pantetheine-containing multienzymes, and the ATP-driven activation of amino acids now appear to be common features of the nonribosomal synthesis of many peptide antibiotics (4-6, 13-1]8).The three complementary enzymes of tyrocidine synthesis (9,12,16) acid activating subuni...

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Cell-Free Synthesis of Gramicidin S^*

Cited by 79 publications

References 23 publications

Nonribosomal biosynthesis of peptide antibiotics

Nonribosomal biosynthesis of peptide antibiotics

Purification and Properties of Branched Chain Amino Acid Aminotransferase from Gramicidin S-Producing Bacillus brevis.

Isolation of amino acid activating subunit-pantetheine protein complexes: Their role in chain elongation in tyrocidine synthesis

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Cell-Free Synthesis of Gramicidin S*

Cited by 79 publications

References 23 publications

Nonribosomal biosynthesis of peptide antibiotics

Nonribosomal biosynthesis of peptide antibiotics

Purification and Properties of Branched Chain Amino Acid Aminotransferase from Gramicidin S-Producing Bacillus brevis.

Isolation of amino acid activating subunit-pantetheine protein complexes: Their role in chain elongation in tyrocidine synthesis

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Cell-Free Synthesis of Gramicidin S^*