The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains

Abstract

The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His 6 -tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PP i exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The K m values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3 end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.The biosynthetic system required for the production of the cyclic decapeptide tyrocidine is one of the prototypes for a protein template-driven pathway to synthesize biologically active peptides by the nonribosomal mechanism (20,21,30,44,46). Large enzymes, called peptide synthetases, activate the amino acid constituents as aminoacyl adenylate at the expense of ATP and thioesterify them on the thiol moiety of an enzyme-attached cofactor, 4Ј-phosphopantetheine. Subsequently, activated amino acids are condensed stepwise in an aminoto carboxy-terminal direction. Previous investigations at the protein chemical level and, in particular, comparisons of several genes encoding peptide synthetases have revealed the modular architecture of this class of enzymes (4,23,28,42,44,53). One module is defined to harbor all catalytic activities to incorporate a single amino acid residue into the peptide product. The modules coincide in number with the number of incorporated amino acids and are arr...

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

“…3) encoding a nonribosomal peptide synthetase (NRPS). The amino acid sequence of CmlP shows strong similarity to sequences of the NRPSs that activate phenylalanine for biosynthesis of gramicidin and tyrocidin (Conti et al, 1997;Mootz & Marahiel, 1997). Analysis of the CmlP sequence detects functional motifs for an adenylation domain with the specificity needed to activate p-aminophenylalanine (PAPA) or p-aminophenylserine (PAPS).…”

Editing of non-cognate aminoacyl adenylates by peptide synthetases

“…The first level of discrimination occurs during substrate recognition through rejection of the non-cognate amino acid by the active site providing a characteristic substrate profile for each adenylate-forming domain. The amino acid-activating domains of the tyrocidinebiosynthesis system can either accurately distinguish between two structurally related substrates or exhibit a relaxed but defined substrate specificity for two amino acids whose incorporation is determined by their relative concentrations [26,27]. Tyrocidine produced by B. bre is is a mixture of four cyclic decapeptides containing tryptophan in place of the phenylalanine and tyrosine residues in positions 3, 4 and 7.…”

Biosynthesis of rapamycin and its regulation: past achievements and recent progress

“…12 The evidence that pipecolate incorporation and macrolactone ring closure are carried out by pipecolate-incorporating enzyme was obtained by a nucleotide sequence analysis and disruption of rapP from S. hygroscopicus. 47 A comparison of the deduced amino-acid sequence of rapP with those of authentic peptide synthetases, such as GrsT from gramicidin S, 48 TycF from tyrocidine 49 and SrfD from surfactin biosynthetic gene cluster, 50 revealed the presence of highly conserved motifs for ATP binding, aminoacyl adenylate formation, peptide bond formation and substrate transfer. 47 When rapP was disrupted from the chromosome of S. hygroscopicus, rapamycin production was significantly reduced.…”