Molecular Cloning and Nucleotide Sequence of the Gramicidin S Synthetase 1 Gene1

Hori, Kazuko; Yamamoto, Yoshihiro; Minetoki, Toshitaka; Kurotsu, Toshitsugu; Kanda, Masayuki; Miura, Setsuko; Okamura, Kaori; Furuyama, Jun-ichi; Saito, Yoshitaka

doi:10.1093/oxfordjournals.jbchem.a122909

Cited by 66 publications

(41 citation statements)

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“…GrsA contains the initiating module of an NRPS involved in the synthesis of gramicidin A (Hori et al, 1989;Krätzschmar et al, 1989). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58

2004

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Using the complete genome sequence from Agrobacterium tumefaciens C58, the authors identified a secondary metabolite gene cluster that encodes the biosynthesis of a metabolite with siderophore activity. Support for this conclusion came from genetic and regulatory analysis of the gene cluster, along with the purification of a metabolite from A. tumefaciens C58 with iron-chelating activity. Genetic analysis of mutant strains disrupted in this gene cluster showed that these strains grew more slowly than the wild-type strain in medium lacking iron. Additionally, the mutant strains failed to produce a chrome-azurol-S-reactive material in liquid or solid medium, and failed to produce the metabolite with iron-chelating characteristics that was identified in the wild-type strain. Addition of this purified metabolite to the growth medium of a mutant strain restored its ability to grow in iron-deficient medium. Furthermore, expression of this gene cluster was induced by growth under iron-limiting conditions, suggesting that expression of this gene cluster occurs when iron is scarce. These data are all consistent with the proposal that the proteins encoded by this gene cluster are involved in the production of a siderophore. Interestingly, these proteins show the highest level of amino acid similarity to proteins from a gene cluster found in the filamentous cyanobacterium Nostoc sp. PCC7120, rather than to known siderophore biosynthetic enzymes. Given these properties, it is proposed that the siderophore produced by A. tumefaciens C58 will have a unique chemical structure. Production of the siderophore was not required for virulence of A. tumefaciens when tested with a standard stem inoculation assay.

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“…GrsA contains the initiating module of an NRPS involved in the synthesis of gramicidin A (Hori et al, 1989;Krätzschmar et al, 1989). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58

2004

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“…A coupling of multifunctional synthase and transferase activities is believed to occur during gramicidin biosynthesis; a thioesterase-like domain, located at the 5Ј end of the gramicidin synthase gene and displaying homology to fatty acid thioesterases, has been suggested to function as a transacylase in the chain termination step (26,27). Similar mechanisms also appear to be involved in the synthesis of nonribosomal peptide synthases such as ␦-(L-␣-aminoadipyl)-Lcysteinyl-D-valine synthase, which is involved in penicillin and cephalosporin biosynthesis (28,29) and the bialaphos antibiotic synthesizing gene cluster (30).…”

Section: Discussionmentioning

confidence: 99%

An Acyl-CoA Synthase (acoas) Gene Adjacent to the Mycocerosic Acid Synthase (mas) Locus Is Necessary for Mycocerosyl Lipid Synthesis in Mycobacterium tuberculosisvar. bovis BCG

Fitzmaurice

Kolattukudy

1998

Journal of Biological Chemistry

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An open reading frame, ORF3, first identified adjacent to the mycocerosic acid synthase gene in Mycobacterium bovis BCG encodes a protein with acyl-CoA synthase (ACoAS) activity. Genes homologous to acoas are found adjacent to other multifunctional polyketide synthase genes in the mycobacterial genome. To test whether these gene products are necessary to esterify the fatty acids generated by the adjacent polyketide synthase gene products, the acoas gene was disrupted in M. bovis BCG using a suicide vector containing the acoas gene with an internal deletion and the hygromycin-resistant gene as selection marker. Allelic exchange at the acoas locus was confirmed by Southern hybridization and polymerase chain reaction amplification of both flanking regions expected from homologous recombination. Immunoblot analysis indicated that the 65-kDa ACoAS protein product was absent in the mutant. Chromatographic analysis of lipids derived from [1-14 C]propionate showed that the mutant did not produce mycocerosyl lipids, although it produced normal levels of mycocerosic acid synthase. These results suggest that ACoAS is involved in the synthesis of mycocerosyl lipids of the mycobacterial cell wall.

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“…Tryptic digestion of gramicidin S synthetase 2, a 512 kDa multienzyme integrating 4 modules, led to amino acidactivating domains of I 10-115 kDa [35][36][37]. The Pro-activating domain has been analysed in detail, subcloned, and can be concluded to be truncated before the J-motif [38]. The 6-(L-~-aminoadipyl)-L-cysteinyl-D-valine synthetase, containing three modules, has been digested by subtilisin, and some fragments have been identified by N-terminal sequencing [5].…”

Section: Discussionmentioning

confidence: 99%

“…Attempts to over-express peptide synthetases or modules for detailed characterization have been carried out with gramicidin S synthetase 1 [38], tyrocidine synthetase 1 [8], the serine-activating enzyme of the enterobactin pathway [41], and the aminoadipate activating module of ACV synthetase [42]. In none of the cases have enzyme activities comparable to the wild-type enzymes been reported concerning aminoacylation, which has been attributed to incomplete post-translational processing.…”

Section: Discussionmentioning

confidence: 99%

Expression of an active adenylate‐forming domain of peptide synthetases corresponding to acyl‐CoA‐synthetases

et al. 1995

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Peptide synthetases and acyl-CoA-synthetases form acyl adenylates which are transferred to CoA or enzyme-bound pantetheine. To verify the existence of an adenylate domain in peptide synthetases, a 60.8 kDa fragment of tyrocidine 1-synthetase was constructed by a 1629 bp deletion, expressed in Escherichia coli, and characterized. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over-expressed synthetase, as judged by ATP-[32p]pp~ exchange reaction. Thus the N-terminal domain resembling an acyl-CoA-synthetase is an autonomous structural element. This N-terminal domain is followed by a cofactor binding domain, resembling acyl carrier proteins involved in polyketide formation.Key words." Tyrocidine synthetase; Multienzyme; Peptide synthetase; Acyl-CoA-synthetase; Pantetheine; CoA structed of modules, each contributing one amino-or hydroxy acid to the peptide structure. Each module contains an adenylate-forming domain, a cofactor binding site, and a condensation/modification domain. The adenylate forming domains display significant similarity to acyl-CoA-synthetases and related proteins. We have predicted, by alignment of sequences of these three classes and those of acyl carrier proteins also binding 4'-phosphopantetheine, the boundaries of adenylate and cofactor domains. The linker region is not rich in Ala, Pro and Glu residues, as in fatty acid synthases and 2-oxo-acid dehydrogenases containing similar cofactor domains. Still, amino acids frequently found in linker regions are dominating. Upon deletion of the cofactor and following functional domains of tyrocidine synthetase 1, a stable N-terminal fragment was obtained which catalysed adenylate formation with similar kinetic properties as the apo-form of the peptide synthetase [8].

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Molecular Cloning and Nucleotide Sequence of the Gramicidin S Synthetase 1 Gene1

Cited by 66 publications

References 0 publications

Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58

Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58

An Acyl-CoA Synthase (acoas) Gene Adjacent to the Mycocerosic Acid Synthase (mas) Locus Is Necessary for Mycocerosyl Lipid Synthesis in Mycobacterium tuberculosisvar. bovis BCG

Expression of an active adenylate‐forming domain of peptide synthetases corresponding to acyl‐CoA‐synthetases

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