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.

Effects of the anthelmintic drug PF1022A on mammalian tissue and cells.

Nematode infections cause human morbidity and enormous economic loss in livestock. Since resistance against currently available anthelmintics is a worldwide problem, there is a continuous need for new compounds. The cyclooctadepsipeptide PF1022A is a novel anthelmintic that binds to the latrophilin-like transmembrane receptor important for pharyngeal pumping in nematodes.

In vitro synthesis of new cyclodepsipeptides of the PF1022-type: probing the alpha-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 alpha-D-hydroxy acids with various structural modifications in the side chain. Thus, new PF1022 derivatives for example, propargyl-D-lactyl-PF1022 and beta-thienyl-D-lactyl-PF1022 were generated.

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 (14)C-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.

New beauveriolides produced by amino acid-supplemented fermentation of Beauveria sp. FO-6979.

Five new beauveriolides were isolated from the acetone extracts of Beauveria sp. FO-6979 mycelia fermented in amino acid-supplemented media. The structures were elucidated by spectroscopic studies including NMR experiments and chemical degradation.

Antimycobacterial activity of lipodepsipeptides produced by Pseudomonas syringae pv syringae B359.

Some lipodepsipeptides produced by Pseudomonas syringae pv. syringae showed strong antimycobacterial activity towards Mycobacterium smegmatis. MIC values found were between 1.

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

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.

Directed biosynthesis of new enniatins.

New cyclohexadepsipeptides of the enniatin type with potential anthelmintic properties were produced by two different strategies: 1. In vitro synthesis by use of the multienzyme enniatin synthetase, and 2. in vivo precursor feeding of enniatin producing strains Fusarium scirpi and Fusarium sambucinum.

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 and S-adenosyl-l-methionine (AdoMet)-dependent N-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 from Fusarium scirpi to gain insight into the assembly of the AdoMet-binding site.

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 precursors L-leucine, D-lactate, D-phenyllactate, and S-adenosyl-L-methionine in the presence of ATP and MgCl(2).

Capsid protein-encoding genes of hamster polyomavirus and properties of the viral capsid.

On the basis of its genome organization the hamster polyomavirus (HaPV) is closely related to the murine polyomavirus Py. But HaPV infection, in contrast to Py infection, gives rise to two different tumor types; depending on the hamster strain used for infection, HaPV induces either epitheliomas or lymphomas. Although the HaPV virions were shown to be similar to those of Py and SV40, more precise information about the structure and protein composition of the HaPV capsid was still missing.

Biosynthesis of taxol: enzymatic acetylation of 10-deacetylbaccatin-III to baccatin-III in crude extracts from roots of Taxus baccata.

The biosynthesis of taxol is a multistep process. One intermediate reaction is the acetylation of 10-deacetylbaccatin-III (10-DAB) to baccatin-III, an assumed precursor of taxol. Here we describe the cell free acetylation of 10-DAB in crude extracts from roots of Taxus baccata saplings using 14C-or 3H-labeled acetyl-coenzyme A as the acetyl donor.

Effect of disruption of the enniatin synthetase gene on the virulence of Fusarium avenaceum.

Production of the phytotoxic compound enniatin has been proposed to play a role during the infection process of plants by enniatin-synthesizing Fusarium species. Enniatins are cyclohexadepsipeptides synthesized by the multifunctional enzyme enniatin synthetase. To test the hypothesis that enniatin contributes to pathogenicity, enniatin-nonproducing mutants were constructed by gene disruption of the enniatin synthetase gene of a virulent Fusarium avenaceum strain.

Enniatin production by fusarium strains and its effect on potato tuber tissue.

Several Fusarium strains produce the cyclohexadepsipeptide enniatin, a host-nonspecific phytotoxin. Enniatins are synthesized by the 347-kDa multifunctional enzyme enniatin synthetase. In the present study, 36 Fusarium strains derived from a wide range of host plants were characterized with respect to enniatin production in different media.

Highly conserved N-methyltransferases as an integral part of peptide synthetases.

The methyltransferase portion of the N-methyl-peptide-synthetase gene, synthesizing enniatin from Fusarium sambucinum, was amplified with the polymerase chain reaction (PCR) using primers derived from the highly conserved sequences of the flanking peptide synthetase domain. The deduced amino acid sequence of the product shares high similarity to the 430 amino acid methyltransferase portion of enniatin synthetase of Fusarium scirpi and the corresponding portions of another fungal peptide synthetase catalyzing the biosynthesis of the N-methylated cyclopeptide cyclosporin. As the methyltransferase portions show only local similarity to motifs apparently conserved within methyltransferases, the segments of peptide synthetases involved in the biosynthesis of bioactive peptides represent a new class of S-adenosyl-L-methionine dependent methyltransferases.

Arrangement of catalytic sites in the multifunctional enzyme enniatin synthetase.

Enniatin synthetase is an N-methyl peptide synthetase comprising 3131 amino acids. Catalytic sites of the 347-kDa multifunctional enzyme were mapped by N-terminal sequencing of substrate affinity-labelled enzyme fragments formed by proteolysis, and functional studies of purified enniatin synthetase fragments. An N-terminal 200-kDa fragment containing the cofactor 4'-phosphopantetheine was able to activate D-hydroxyisovaleric acid (D-HOiVl) as a thioester.

Cloning and sequencing of a cyclophilin gene from the cyclosporin producer Tolypocladium niveum.

The gene of a 19 kDa cyclophilin of Tolypocladium niveum was isolated and sequenced. The open reading frame of 956 bp is interrupted by four introns of 76, 221, 58, and 61 bp size. 101 nucleotides upstream of the start codon ATG we found a putative promoter region containing a TATA and a CAAT box.

Bacterial expression of catalytically active fragments of the multifunctional enzyme enniatin synthetase.

Enniatin synthetase catalyzes the biosynthesis of N-methylated cyclohexadepsipeptides. The 347 kDa enzyme is encoded by the esyn1 gene of Fusarium scirpi and contains two domains (EA and EB) homologous to each other and to regions of other microbial peptide synthetases. Parts of the esyn1 gene were subcloned in frame to a small lacZ gene portion of Escherichia coli expression vectors.

Purification and characterization of eucaryotic alanine racemase acting as key enzyme in cyclosporin biosynthesis.

A specific alanine racemase, which is a key enzyme in the biosynthesis of the undecapeptide cyclosporin A, was purified to electrophoretic homogeneity from the fungus Tolypocladium niveum. This is the first enzyme of this kind isolated from a eucaryotic organism. The enzyme catalyzes the reversible racemization of alanine and requires pyridoxal phosphate as the exclusive cofactor.

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 (M(r) 346,900).