Till Hornbogen

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.

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.

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.

Functional Characterization of the Recombinant N-Methyltransferase Domain from the Multienzyme Enniatin Synthetase

A 51 kDa fusion protein incorporating the N‐methyltransferase domain of the multienzyme enniatin synthetase from Fusarium scirpi was expressed in Saccharomyces cerevisiae. The protein was purified and found to bind S‐adenosyl methionine (AdoMet) as demonstrated by cross‐linking experiments with 14C‐methyl‐AdoMet under UV irradiation. Cofactor binding at equilibrium conditions was followed by saturation transfer difference (STD) NMR spectroscopy, and the native conformation of the methyltransferase was assigned. STD NMR spectroscopy yielded significant signals for H2 and H8 of the adenine moiety, H1’ of D‐ribose, and SCH3 group of AdoMet. Methyl group transfer catalyzed by the enzyme was demonstrated by using aminoacyl‐N‐acetylcysteamine thioesters (aminoacyl‐SNACs) of L‐Val, L‐Ile, and L‐Leu, which mimic the natural substrate amino acids of enniatin synthetase presented by the enzyme bound 4′‐phosphopantetheine arm. In these experiments the enzyme was incubated in the presence of the corresponding aminoacyl‐SNAC and 14C‐methyl‐AdoMet for various lengths of time, for up to 30 min. N‐[14C‐Methyl]‐aminoacyl‐SNAC products were extracted with EtOAc and separated by TLC. Acid hydrolysis of the isolated labeled compounds yielded the corresponding N‐[14C‐methyl] amino acids. Further proof for the formation of N‐14C‐methyl‐aminoacyl‐SNACs came from MALDI‐TOF mass spectrometry which yielded 23 212 Da for N‐methyl‐valyl‐SNAC, accompanied by the expected postsource decay (PSD) pattern. Interestingly, L‐Phe, which is not a substrate amino acid of enniatin synthetase, also proved to be a methyl group acceptor. D‐Val was not accepted as a substrate; this indicates selectivity for the L isomer.

Biosynthesis of Depsipeptide Mycotoxins in Fusarium

The cyclic hexadepsipeptide enniatin is known as a phytopathogenic compound from Fusaria causing necrosis and wilt. The molecule consists of three alternating residues each of a branched chain amino acid and D-hydroxyisovaleric acid (D-Hiv). Enniatins are synthesized by a 347kDa multienzyme (enniatin synthetase) via a thiol template mechanism. The corresponding gene esyn1 has an open reading frame of 9393 nucleotides and harbours two modules, one responsible for D-hydroxy acid activation and one for L-amino acid activation with an integrated N-methyltransferase domain. Such methyltransferases build an homologous group among N-methyl peptide synthetases. Enniatins are synthesized by step-wise condensation of dipeptidol building blocks in an iterative manner resembling fatty acid synthesis. A key enzyme in enniatin biosynthesis is the NADPH-dependent D-2-hydroxyisovalerate dehydrogenase, that supplies enniatin synthetase with D-Hiv. Enniatins contribute to the wilt toxic character of Fusaria. Virulence was significantly reduced in F. avenaceum after disruption of the esyn1 gene.

Two new cyclophilins from Fusarium sambucinum and Aspergillus niger: Resistance of cyclophilin/cyclosporin a complexes against proteolysis

Two new peptidyl-prolyl-cis/trans-isomerases were purified to homogeneity from Fusarium sambucinum and Aspergillus niger. They belong to the class of cyclosporin A binding proteins (cyclophilins) and have molecular masses of about 18 kDa. As has been shown for other cyclophilins, the isomerase activity of the enzymes is inhibited by cyclosporin A in the nanomolar range. Furthermore binding of cyclosporin A prevents proteolytic digestion of the cyclophilin/cyclosporin complexes by the endoproteases GluC, LysC and alpha-chymotrypsin, in contrast to the free cyclophilins, which are readily cleaved by these proteases. We could also observe this protection for cyclophilins from sheep thymus and from the cyclosporin producing fungus Tolypocladium inflatum.

The Fatty Acid Synthase of the Basidiomycete Omphalotus olearius Is a Single Polypeptide

Fatty acids are essential components of almost all biological membranes. Additionally, they are important in energy storage, as second messengers during signal transduction, and in post-translational protein modification. De novo synthesis of fatty acids is essential for almost all organisms, and entails the iterative elongation of the growing fatty acid chain through a set of reactions conserved in all kingdoms. During our work on the biosynthesis of secondary metabolites, a 450-kDa protein was detected by SDS-PAGE of enriched fractions from mycelial lysates from the basidiomycete Omphalotus olearius. Protein sequencing of this protein band revealed the presence of peptides with homology to both α and β subunits of the ascomycete fatty acid synthase (FAS) family. The FAS encoding gene of O. olearius was sequenced. The positions of its predicted 21 introns were verified. The gene encodes a 3931 amino acids single protein, with an equivalent of the ascomycetous β subunit at the N-terminus and the α subunit at the C-terminus. This is the first report on an FAS protein from a homobasidiomycete and also the first fungal FAS which is comprised of a single polypeptide.