Cloning of the tyrocidine synthetase 1 gene from Bacillus brevis and its expression in Escherichia coli

Marahiel, Mohamed A.; Krause, Michael; Skarpeid, Hans‐Jacob

doi:10.1007/bf00425664

Cited by 47 publications

(34 citation statements)

References 31 publications

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“…The grsA gene encodes a protein of 126,631 Da, which is in agreement with the apparent molecular mass of 120,000 Da previously determined for the gramicidin S synthetase 1 (29,45). The DNA sequence is further supported by the coincidence of its predicted amino acid sequence with the recently determined amino-terminal sequence (10 residues) of the gramicidin S synthetase 1 purified from B. brevis (Vater et al, in press).…”

Section: Methodssupporting

confidence: 85%

“…The activated amino acid is transferred to a thiol group (a cysteine residue) on the enzyme, yielding a covalently bound thioester-linked amino acid (25). The two enzymes have similar molecular masses and cross-react immunochemically (29). To evaluate the possibility that the two enzymes might have a common evolutionary ancestry or have been generated by gene duplication, we compared their predicted amino acid sequences.…”

Section: Methodsmentioning

confidence: 99%

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Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases

1989

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The DNA sequence of about 5.9 kilobase pairs (kbp) of the gramicidin S biosynthesis operon (grs) was determined. Three open reading frames were identified; the corresponding genes, cafled grsT, grsA, and grsB, were found to be organized in one transcriptional unit, not two as previously reported (M. Krause and M. A. Marahiel, J. Bacteriol. 170:46694674, 1988). The entire nucleotide sequence of grsA, coding for the 126.663-kilodalton gramicidin S synthetase 1, grsT, encoding a 29.191-kilodalton protein of unknown function, and 732 bp of the 5' end of grsB, encoding the gramicidin S synthetase 2, were determind. A dngle initiation site of transcription 81 bp upstream of the grsT initiation codon GTG was identified by high-resolution S1 mapping studies. The sequence of the grsA gene product showed a high degree of homoogy to the tyrocidine synthetase 1 (TycA protein), and that of grsT exhibited a signUicant degree of homology to vertebrate fatty acid thioesterases.In response to certain nutrient conditions usually associated with nutrient depletion, gram-positive bacteria of the genus Bacillus induce the process of endospore formation as well as the production of secondary metabolites such as peptide antibiotics and extracellular proteases (4, 26, 41). In both cases, genes are activated at the transition from logarithmic to stationary phase of growth. Expression of these genes depends on the products of spoO loci (10,26,30,41). To understand the complex relationship between induction of sporulation'and production of secondary metabolites, we and others have isolated and studied the organization and regulation of some antibiotic biosynthesis genes at the molecular level. Genes encoding multifunctional enzymes involved in biosynthesis of the cyclic antibiotics bacitracin, gramicidin S, and tyrocidine have been identified (16,20,33). The nonribosomal synthesis of these antibiotics by the so-called protein-thiotemplate mechanism has been studied extensively (18,22,23,25). In analogy with fatty acid synthetase, the cofactor 4'-phosphopantetheine is involved in translocation of the growing polypeptide chain through transthiolation. However, there is one important difference: the fatty acid chain is synthesized by condensation of identical carbon units, whereas in polypeptide synthesis, different amino acids are polymerized in a defined sequence given by the position of the corresponding domain on the multienzyme.Recently, we reported that the biosynthesis genes for gramicidin S, grsA and grsB, and those involved in tyrocidine biosynthesis, tycA and tycB, are clustered (20,33 gland and duck uropygial gland (38, 39). In addition, Si nuclease protection studies located a single transcription initiation site upstream of the grsT OREF, which may control the expression of the entire antibiotic biosynthesis operon in vivo. MATERIALS AND METHODSBacterial strains and growth coditions. Escherichia coli JM105 and JM83 (32) we're used as hosts for the recombinant plasmids and were grown in L-broth medium supplemented with the appro...

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Section: Methodssupporting

confidence: 85%

Section: Methodsmentioning

confidence: 99%

Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases

1989

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“…The plasmid pGC12 contains a 3.5 kb HinclI fragment (containing the TYI coding region) from pBT2 [13] and carries the ampicillin resistence gene and a portion of the lac operon, including the lac promoter, operator and part of the Z gene. Plasmid DNA was isolated by the alkali method of Birnboim and Doly [14].…”

Section: Isolation Of Plasmid Dnamentioning

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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“…We have chosen tyrocidine synthetase I with specificity for phenylalanine (2, 4) and the valine-activating module of surfactin synthetase as models (15,30). Both modules are well characterized and can be overexpressed and isolated as active proteins from the heterologous host Escherichia coli (2,24,27). Variable regions of the two modules with different substrate specificities were combined reciprocally by genetic recombination.…”

mentioning

confidence: 99%

“…Tyrocidine synthetase and surfactin synthetase are multifunctional peptide synthetases produced by Bacillus brevis and Bacillus subtilis, respectively (1)(2)(3)(4)(5). The enzymes belong to a superfamily of adenylate-forming enzymes (6 -8).…”

mentioning

confidence: 99%

Substrate Specificity of Hybrid Modules from Peptide Synthetases

Elsner

Engert

Hamoen

et al. 1997

Journal of Biological Chemistry

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Homologous modules from two different peptide synthetases were analyzed for functionally equivalent regions. Hybrids between the coding regions of the phenylalanine-activating module of tyrocidine synthetase and the valine-activating module of surfactin synthetase were constructed by combining the two reading frames at various highly conserved consensus sequences. The resulting DNA fragments were expressed in Escherichia coli as C-terminal fusions to the gene encoding for the maltose-binding protein. The fusion proteins were purified, and the amino acid specificities, the acceptance of different nucleotide analogues, and the substrate binding affinities were analyzed. We found evidence for a large N-terminal domain and a short C-terminal domain of about 19 kDa within the two modules, which are separated by the sequence motif GELCIGG. The two domains could be reciprocally transferred between the two modules, and the constructed hybrid proteins showed amino acid adenylating activity. Hybrid proteins fused at various consensus motifs within the two domains were inactive, indicating that the domains may fold independently and represent complex functional units. The N-terminal domain was found to be responsible for the amino acid specificity of the modules, and it is also involved in the recognition of the ribosyl and the phosphate moieties of the nucleotide substrate. For tyrocidine synthetase I, we could confine the sites for amino acid specificity to a region of 330 residues. The C-terminal domain is essential for the enzymatic activity and has a strong impact on the specific activity of the modules.Tyrocidine synthetase and surfactin synthetase are multifunctional peptide synthetases produced by Bacillus brevis and Bacillus subtilis, respectively (1-5). The enzymes belong to a superfamily of adenylate-forming enzymes (6 -8). Small peptides like tyrocidine A and surfactin are formed by a nonribosomal pathway according to the thio-template mechanism (6, 9). Prior to incorporation, amino acids and related compounds are activated as adenylates by cleavage of ATP and release of pyrophosphate. Peptide synthetases exhibit a modular structure with several linked modules of about 100 kDa (1, 10 -16). Each module is responsible for recognition, activation, and incorporation of a specific amino acid constituent into the peptide product. Various modules of peptide synthetases have been sequenced, and several highly conserved motifs were found (7,8,11,14,(17)(18)(19). Mutagenesis and cross-linking experiments gave evidence for the involvement of most of these motifs in the binding of ATP and the adenylate forming activity (20 -23). A serine residue in the highly conserved sequence motif LGG(H/D)S at the C terminus of the modules was clearly identified as the site for covalent attachment of a phosphopantetheine cofactor (24 -26). The deletion of the cofactor attachment site in the phenylalanine-activating modules of tyrocidine synthetase I and gramicidin S synthetase I did not affect the amino acid adenylate forming activity (...

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Cloning of the tyrocidine synthetase 1 gene from Bacillus brevis and its expression in Escherichia coli

Cited by 47 publications

References 31 publications

Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases

Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases

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

Substrate Specificity of Hybrid Modules from Peptide Synthetases

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