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 alpha and beta 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 beta subunit at the N-terminus and the a 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.

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. STD NMR spectroscopy yielded significant signals for H(2) and H(8) of the adenine moiety, H(1') of D-ribose, and S-CH(3) 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 (14)C-methyl-AdoMet for various lengths of time, for up to 30 min. N-[(14)C-Methyl]-aminoacyl-SNAC products were extracted with EtOAc and separated by TLC. Acid hydrolysis of the isolated labeled compounds yielded the corresponding N-[(14)C-methyl] amino acids. Further proof for the formation of N-(14)C-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.

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. 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 [(14)C]-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 (S(20,w)=14.1S) than expected (S(20,w)=16.9S) for a globular protein, indicating that Esyn has an extended structure.