D-Lysergyl Peptide Synthetase from the Ergot Fungus Claviceps purpurea

Riederer, Brigitte; Han, Mehmet Vefa; Keller, Ullrich

doi:10.1074/jbc.271.44.27524

Cited by 77 publications

(71 citation statements)

References 43 publications

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“…They are comparable to those determined for wild-type GrsB, the most closely related peptide synthetase (18). The K m values obtained are also in the same order of magnitude as those reported for other peptide synthetases (8,33,45) and for aminoacyl-tRNA synthetases. The PheA4 and TyrA7 domains did indeed have higher affinities to L-Trp (K m values of 0.08 and 0.10 mM, respectively) than to L-Phe (0.45 mM) and L-Tyr (0.30 mM), respectively.…”

Section: Resultssupporting

confidence: 73%

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

1997

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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...

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Section: Resultssupporting

confidence: 73%

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

1997

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“…and bacilli, as well as animals and humans (14,16,22). DKPs can be formed after proteolytic digestion of precursor molecules or synthesized from dedicated NRPS systems, as was shown for the biosynthetic clusters for ergotamine (36) and albonoursin (22). Thus, our engineered TycA/TycB1 system may serve as a starting point for the production of engineered variants of this class of compounds (16).…”

Section: Discussionmentioning

confidence: 99%

In Vivo Production of Artificial Nonribosomal Peptide Products in the Heterologous Host Escherichia coli

Gruenewald

Mootz

Stehmeier

et al. 2004

Appl Environ Microbiol

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Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin. Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches. Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule. In the present study, we describe the fermentative production of the cyclic dipeptide D-Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host Escherichia coli, and the use of artificial nonribosomal peptide synthetases. E. coli provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4-phosphopantetheine transferase gene sfp to ensure heterologous production of fully active holoenzmyes. D-Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in Bacillus brevis. Coexpression of the corresponding genes in E. coli gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment. Furthermore, the cyclic dipeptide is stable and not toxic to E. coli and is secreted into the culture medium without the need for any additional factors. Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature. By these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of D-Phe-Pro-diketopiperazine/liter. As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals.

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“…Another well-characterized step is the incorporation of lysergic acid into D-lysergyl peptide lactams, oligopeptide precursors of ergotamine, and related alkaloids. This step is carried out by a complex of two peptide synthetases recently purified from C. puvpurea (Riederer et al, 1996). It is likely that a closely related enzyme complex is involved in ergovaline biosynthesis in Epichloe and Neotyphodium species.…”

Section: Biosynthesismentioning

confidence: 99%