The fungus Trichoderma virens is a ubiquitous soil saprophyte that has been applied as a biological control agent to protect plants from fungal pathogens. One mechanism of biocontrol is mycoparasitism, and T. virens produces antifungal compounds to assist in killing its fungal targets. Peptide synthetases produce a wide variety of peptide secondary metabolites in bacteria and fungi. Many of these are known to possess antibiotic activities. Peptaibols form a class of antibiotics known for their high ␣-aminoisobutyric acid content and their synthesis as a mixture of isoforms ranging from 7 to 20 amino acids in length. Here we report preliminary characterization of a 62.8-kb continuous open reading frame encoding a peptaibol synthetase from T. virens. The predicted protein structure consists of 18 peptide synthetase modules with additional modifying domains at the N-and C-termini. T. virens was shown to produce a mixture of peptaibols, with the largest peptides being 18 residues. Mutation of the gene eliminated production of all peptaibol isoforms. Identification of the gene responsible for peptaibol production will facilitate studies of the structure and function of peptaibol antibiotics and their contribution to biocontrol activity.
Trichogin A IV (GA IV) is the main component of the natural trichogin mixture, a new peptide group extracted from in vitro cultures of the fungus Trichoderma longibrachiatum. G A IV was isolated by reversed-phase HPLC, and its amino acid sequence was elucidated by FAB mass spectrometry and high-field NMR. Complete IH and I3C resonance assignments were carried out using HOHAHA, ROESY, 'H-I3C COSY, and COLOC two-dimensional spectroscopies. This linear peptide contains an N-terminal extremity acylated by an octanoyl group, 10 amino acids, and a leucinol C-terminal amino alcohol, giving rise to a novel class of peptides we propose to name lipopeptaibols. The methanolic solution conformation of G A IV was examined by a combination of CD data, "C N M R relaxation measurements, temperature coefficients of NH and CO groups, and N O E data. The structure was found to be helical. The membrane-modifying properties were tested toward liposomes composed of egg phosphatidylcholine with 20 or 30% cholesterol. GA IV revealed permeability modifications similar to those exhibited by a 19-residue acidic peptaibol.
Trichogin GA IV, an 11-residue lipopeptaibol blocked at
the N-terminus by an n-octanoyl group and at
the C-terminus by a 1,2-amino alcohol (l-leucinol),
extracted from the fungus Trichoderma
longibrachiatum,
exhibits
remarkable membrane-modifying properties. We have synthesized
trichogin GA IV and several
[l-Leu-OMe11]
analogs carrying at the N-terminus an acyl chain of variable length
(C2−C8, C10, C12,
C14, C16, C18). A
succinoylated
head-to-head dimer was also prepared. A conformational analysis,
carried out by FTIR absorption, CD, and NMR,
showed that the right-handed helical structure of the natural
lipopeptaibol is essentially preserved in all its analogs.
Permeability measurements revealed that at least six carbon atoms
in the Nα-blocking fatty acyl moiety are
required
for the onset of significant membrane-modifying properties. Also
the head-to-head dimer is remarkably active.
Possible models for the mechanism of membrane permeability of
trichogin GA IV are discussed.
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