Rainer Zocher

Molecular characterization of the enniatin synthetase gene encoding a multifunctional enzyme catalysing N-methyldepsipeptide formation in Fusarium scirpi

The gene encoding the multifunctional enzyme enniatin synthetase from Fusarium scirpi (esyn1) was isolated and characterized by transcriptional mapping and expression studies in Escherichia coli. This is the first example of a gene encoding an N‐methyl peptide synthetase. The nucleotide sequence revealed an open reading frame of 9393 bp encoding a protein of 3131 amino acids (Mr 346 900). Two domains designated EA and EB within the protein were identified which share similarity to each other and to microbial peptide synthetase domains. In contrast to the N‐terminal domain EA, the carboxyl terminal domain EB is interrupted by a 434‐amino‐acid portion which shows local similarity to a motif apparently conserved within adenine and cytosine RNA and DNA methyltransferases and therefore seems to harbour the N‐methyl‐transferase function of the multienzyme.

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.

In Vitro Synthesis of New Enniatins: Probing the α-D-Hydroxy Carboxylic Acid Binding Pocket of the Multienzyme Enniatin Synthetase

Cyclodepsipeptides are an important and interesting group of natural products. Their bioactivity spans from antitumour, antibiotic, anthelmintic, antifungal, immunosuppressant, antimalaria to anti-inflammatory activities ; therefore they are of great interest for pharmaceutical applications. Particularly important representatives are the 18-membered enniatins (Scheme 1) and the structurally related beauvericins. These have been found in a variety of fungal strains, for example, of the genera Fusarium, Beauveria, Alternaria, Verticillium and Paecilomyces. Enniatins have an alternating arrangement of amide and ester bonds. They consist of three dipeptidol units, comprising a branched chain N-methyl-lamino acid and a-d-hydroxyisovaleric acid (d-Hiv; Scheme 1). 4] They mainly differ in the hydrophobic side chain of the a-l-2-amino acids (enniatin A–O). The d-Hiv moiety is conserved in most enniatins (Scheme 1) and is replaced only in a few cases by a-l-2-hydroxy-3-methylpentanoic acid (enniatin H, I) and 2,3-dihydroxy-3-methylpentanoic acid (enniatin L, M, N). 6] In nonribosomal peptide synthesis the substrates are activated by specific modules that consist of domains, which catalyze condensation, adenylation and thiolation reactions. We have established an in vitro system for the synthesis of enniatins using the nonribosomal peptide synthetase (NRPS) enniatin synthetase (ESyn). This NRPS can be considered as a hybrid system between a peptide synthetase and Nmethyltransferase. It consists of two modules, EA and EB; EA is responsible for a-d-hydroxy acid activation and EB for amino-acid activation and N-methylation. Both modules activate their substrates as pantetheine-derived thioesters with the corresponding acyl adenylates. The process of substrate activation starts with the recognition step by the adenylation domains of modules EA and EB. The specificity of these domains is responsible for the selection of amino and hydroxy acids incorporated into the depsipeptide ring. Enniatins are synthesized in an iterative process by forming intermediate dipeptidol building blocks that are finally condensed head-to-tail to form the cyclohexadepsipeptide. In this process a third protein-bound pantetheine group acts as a “waiting position” for the growing peptide chain.

Thiol Template Peptide Synthesis Systems in Bacteria and Fungi

Publisher Summary This chapter discusses the thiol template peptide synthesis systems in bacteria and fungi. Peptide synthetase systems are defined as the arrangement of various amino-acid activation domains in the form of a multi-enzyme or a multi-enzyme complex. This has been shown by comparisons of various enzyme systems with their corresponding genes. The order of the various activation domains is mirrored in the sequence of the peptide synthesized in prokaryotes. Eukaryotic peptide synthetases always consist of a single polypeptide chain encoded by an intronless gene. This single polypeptide chain harbours the various adenylate formation domains, thioester, and additional modules necessary for the synthesis of a given product. In synthetases from fungi, the peptide is assembled by aminoadipyl-cysteinyl-D-valine synthase (ACVS), which has been isolated from a representative number of fungi and bacteria. Based on biochemical investigations in the cases of the enzymes from A. nidulans and S. clavuligerus and also considering the sequences of a number of ACVS genes, it is clear that this enzyme is composed of three peptide synthetase domains lying on one polypeptide chain of 420 kDa. Much progress in the enzymology of prokaryotic peptide synthetases has been achieved in the field of the acyl peptide lactone synthetases. Acyl peptide lactones consist of peptide lactone rings to which are attached aromatic or aliphatic side groups in an amide-like fashion.

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.

Enniatin synthetase is a monomer with extended structure: evidence for an intramolecular reaction mechanism

Abstract. Enniatin synthetase (Esyn), a 347-kDa multienzyme consisting of two substrate activation modules, is responsible for the nonribosomal formation of the cyclohexadepsipeptide enniatin. The synthesis follows the so-called thiol template mechanism. While this process is basically well established, no substantial insight into the 3-dimensional arrangement of these enzymes and possible interactions between them exists to date. To find out whether enniatin synthesis is an intramolecular process or the result of three interacting Esyn molecules (intermolecular), analytical ultracentrifugation equilibration studies were carried out. The molecular mass of Esyn was determined by ultracentrifugation and is in good agreement with that calculated from the ORF of the encoding gene, indicating that Esyn exists in solution as a monomer. This strongly suggests that synthesis of the cyclohexadepsipeptide enniatin follows an intramolecular reaction mechanism in which all three reaction cycles are catalyzed by a single Esyn molecule. This finding was supported by in vitro complementation studies in which [14C]-methylvalyl Esyn, upon incubation with the second substrate D-2-hydroxyisovaleric acid (D-Hiv) and ATP, did not yield radioactive enniatin. This confirms our previous assumption of an iterative reaction mechanism similar to that for fatty acid synthase. Furthermore, the sedimentation rate constant evaluated from analytical ultracentrifugation was lower (S20,w=14.1S) than expected (S20,w=16.9S) for a globular protein, indicating that Esyn has an extended structure.

In vitro Synthesis of New Cyclodepsipeptides of the PF1022-Type: Probing the α-D-Hydroxy Acid Tolerance of PF1022 Synthetase

The nonribosomal peptide synthetase PF1022‐synthetase (PFSYN) synthesises the cyclooctadepsipeptide PF1022 from the building blocks D‐lactate, D‐phenyllactate and N‐methylleucine. The substrate tolerance of PFSYN for hydroxy acids was probed by in vitro screening of a set of aliphatic and aromatic α‐D‐hydroxy acids with various structural modifications in the side chain. Thus, new PF1022 derivatives for example, propargyl‐D‐lactyl‐PF1022 and β‐thienyl‐D‐lactyl‐PF1022 were generated. The promiscuous behaviour of PFSYN towards aliphatic and aromatic α‐D‐hydroxy acids is considerably larger than that of related enniatin synthetase (ESYN) and thus gives rise to the enzymatic generation of various new PF1022 derivatives.

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.

Mutational Analysis of the N-Methyltransferase Domain of the Multifunctional Enzyme Enniatin Synthetase

N-Methylcyclopeptides like cyclosporins and enniatins are synthesized by multifunctional enzymes representing hybrid systems of peptide synthetases andS-adenosyl-l-methionine (AdoMet)-dependentN-methyltransferases. The latter constitute a new family of N-methyltransferases sharing high homology within procaryotes and eucaryotes. Here we describe the mutational analysis of the N-methyltransferase domain of enniatin synthetase fromFusarium scirpi to gain insight into the assembly of the AdoMet-binding site. The role of four conserved motifs (I,2085VLEIGTGSGMIL; II/Y, 2105SYVGLDPS; IV,2152DLVVFNSVVQYFTPPEYL; and V,2194ATNGHFLAARA) in cofactor binding as measured by photolabeling was studied. Deletion of the first 21 N-terminal amino acid residues of the N-methyltransferase domain did not affect AdoMet binding. Further shortening close to motif I resulted in loss of binding activity. Truncation of 38 amino acids from the C terminus and also internal deletions containing motif V led to complete loss of AdoMet-binding activity. Point mutations converting the conserved Tyr223 (corresponding to position 2106 in enniatin synthetase) in motif II/Y (close to motif I) into Val, Ala, and Ser, respectively, strongly diminished AdoMet binding, whereas conversion of this residue to Phe restored AdoMet-binding activity to ∼70%, indicating that Tyr223 is important for AdoMet binding and that the aromatic Tyr223 may be crucial for AdoMet binding in N-methylpeptide synthetases.

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.