Horst Kleinkauf

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

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]PPi 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.

Biosynthesis of PF1022A and Related Cyclooctadepsipeptides

PF1022A belongs to a recently identified class of N-methylated cyclooctadepsipeptides (CODPs) with strong anthelmintic properties. Described here is the cell-free synthesis of this CODP and related structures, as well as the purification and enzymatic characterization of the responsible synthetase. For PF1022A synthesis extracts of Mycelia sterilia were incubated with the precursorsl-leucine, d-lactate,d-phenyllactate, and S-adenosyl-l-methionine in the presence of ATP and MgCl2. A 350-kDa depsipeptide synthetase, PFSYN, responsible for PF1022A synthesis was purified to electrophoretic homogeneity. Like other peptide synthetases, PFSYN follows a thiotemplate mechanism in which the substrates are activated as thioesters via adenylation. N-Methylation of the substrate l-leucine takes place after covalent binding prior to peptide bond formation. The enzyme is capable of synthesizing all known natural cyclooctadepsipeptides of the PF1022 type (A, B, C, and D) differing in the content of d-lactate andd-phenyllactate. In addition to PF1022 types A, B, C, and D, the in vitro incubations produced PF1022F (a CODP consisting of d-lactate andN-methyl-l-leucine), as well as di-, tetra-, and hexa-PF1022 homologs. PFSYN strongly resembles the well documented enniatin synthetase in size and mechanism. Our results suggest that PFSYN, like enniatin synthetase, is an enzyme with two peptide synthetase domains and forms CODP by repeated condensation of dipeptidol building blocks. Due to the low specificity of thed-hydroxy acid binding site, d-lactate ord-phenyllactate can be incorporated into the dipeptidols depending on the concentration of these substrates in the reaction mixture.

Biosynthesis of cyclosporin A

Abstract Short term feeding of the mould Tolypocladium inflatum with 14C-labelled amino acids revealed a selective incorporation of l -leucine, l -valine, glycine and d , l -alanine into cyclosporins A and C. Feeding of l -[Me-14C]methionine exclusively labelled the N-methyl moieties of the cyclosporins. The distribution of radioactivity from this substrate was directly proportional to the number of the relevant N-methyl amino acids in cyclosporin A, indicating a simultaneous methylation of these residues.

Cyclosporin synthetase is a 1.4 MDa multienzyme polypeptide Re‐evaluation of the molecular mass of various peptide synthetases

The earlier determined molecular mass of 0.8 MDa for the multifunctional polypeptide, cyclosporin synthetase, was re‐evaluated by SDS‐PAGE and CsCl density gradient centrifugation. In SDS‐PAGE, new molecular mass values as standards were available from sequencing data. In the CsCl density gradient extremly low protein concentrations, such as 10–50 nM could be analysed due to the fluorescence detection system of the analytical ultracentrifuge. Both methods yielded approximately the same value of about 1.4 MDa. Using this molecular mass of cyclosporin synthetase as a reference the molecular masses of various related enzymes could be re‐evaluated in SDS‐PAGE. The sedimentation coefficient of 26.3 S for cyclosporin synthetase indicates an oblate overall shape of the enzyme.

Peptide Antibiotics, β-Lactams, and Related Compounds

In the field of natural peptides, beta-lactams, and related compounds, recent exciting developments are discussed. The increasing interest in this class of bioactive amino-acid derived structures has been attributed to the use of new directed screens (enzyme inhibition assays, beta-lactam detection, immunomodulator studies), new and improved applications (antibiotic, transplantation, and cancer chemotherapy), and advances in functional studies (DNA binding peptides, nucleotide complexones, cell wall and protein processing inhibitors). Peptides offer unique access to modifications and analog production by in vivo (directed biosynthesis) and in vitro procedures (enzymatic synthesis) due to their general linear precursors permitting point replacements. Of special interest are recent developments in the genetics of these compounds (cyclic peptides and beta-lactams), which will find applications in production methods in the near future.

Biosynthesis of enniatin B: Partial purification and characterization of the synthesizing enzyme and studies of the biosynthesis

Abstract Enniatin B-synthetase was purified 50-fold from Fusarium oxysporum strain ETH 1536/9. The biosynthesis of this depsipeptide seems to occur in a similar way to that of a number of peptide antibiotics like gramicidin S, tyrocidin and bacitracin. It has been shown that the single precursors of the molecule are activated in the form of thioesters via acyl adenylates. Further evidence will be presented, that N-methyl valine thioester bound to the enzyme is an obligatory intermediate in the biosynthetic process.

Tyrocidine and the linear gramicidin: Do these peptide antibiotics play an antagonistic regulative role in sporulation?

1. The cyclic peptide antibiotic tyrocidine, synthesized by Bacillus brevis (ATCC 8185), inhibits RNA synthesis in an in vitro transcriptional system by forming a complex with the DNA. 2. The linear peptide antibiotic gramicidin, synthesized by the same strain, reverses at least partly this inhibition. The molecular mechanism of this reactivation is unknown. Gramicidin by itself inhibits transcription in vitro. This inhibition is not due to a complex formation between DNA and the peptide. 4. A possible regulative role of the two peptides in sporulation is discussed.

Biosynthesis of Ergotamine in Protoplasts of Claviceps purpurea

Protoplasts of Claviceps purpurea (ATCC 20102) were prepared in 0.8 m-sucrose containing 10 mm-CaCl2 and 10 mm-MgCl2. Protoplasts could revert to the filamentous state but not after treatment with water. Most of the protoplasts (about 80%) were highly vacuolated and these were separated from the non-vacuolated protoplasts and cell debris on the basis of their low density. Only the vacuolated protoplasts were able to synthesize ergotamine and ergocryptine de novo. Protoplasts were about 50% less active than the control mycelium. The control mycelium was more active in the uptake of labelled precursors than both protoplasts and freshly harvested mycelium. In the amino acid pool of protoplasts, alanine was present in a concentration which exceeded that of proline by a factor of six and that of phenylalanine by a factor of 100. This finding is consistent with the incorporation ratios of these amino acids into ergotamine when isotope dilution of the added radiolabel is considered. A significant stimulation of incorporation of constituent amino acids into ergotamine and ergocryptine occurred when d-lysergic acid was added to protoplasts and mycelium.

Alamethicin biosynthesis. Acetylation of the amino terminus and attachment of phenylalaninol

Alamethicin synthetase was extracted from the fungus Trichoderma viride at the end of its exponential growth phase. It is multienzyme complex with a molecular weight of approx. 480 000. The biosynthesis of alamethicin is initiated on the synthetase by acetylation of thiolester-bound aminoisobutyric acid, which remains enzyme bound. Acetyl-CoA serves as the acetate donor. Of the alamethicin constituents, glycine, alanine and valine are also acetylated when incubated alone. This acetylation is prevented by added aminoisobutyric acid, which indicates that the site on alamethicin synthetase catalyzing the acetylation has a preference for aminoisobutyric acid. Alamethicin formation on the synthetase is terminated by linkage of phenylalaninol to the carboxyl terminus of the peptide. It is unlikely that the amino alcohol is a degradation product of alamethicin or that it had been split off from the synthetase complex. Thus it is probably the reaction product of a separate enzyme system.

Enniatin production by Fusarium oxysporum in chemically defined media

SummaryProduction of the depsipeptide antibiotic enniatin was studied in submerged cultures of Fusarium oxysporum. The influence of various nutritional factors on growth and enniatin production was investigated. Sucrose and glucose were suitable carbon sources, NaNO3 was the best nitrogen source with respect to growth and production. Enniatin yields up to 2.5 g/l were achieved within 4 days of cultivation of a variant obtained after two subsequent mutagenic treatments. Addition of several amino acids to producing cultures enhanced product formation up to 5 g/l. External supply of the branched-chain amino acids, valine, leucine, and isoleucine triggered an increased formation of the corresponding enniatin homologues which contain the amino acid fed as a constituent.