By combination of several 1H NMR techniques, the sequence of actagardine has been elucidated. It has been shown that it is a tetracyclic 19-residue peptide antibiotic. It differs from the previously described lanthionine-containing peptide antibiotics belonging to the lantibiotic class.

The three-dimensional solution structure of the lantibiotic actagardine was determined at high resolution by homonuclear and heteronuclear two-dimensional and three-dimensional NMR spectroscopy in [2H3]acetonitrile/H2O (7:3). 133 non-trivial distance and 22 torsional-angle constraints were derived from the NMR data. An ensemble of 15 low-energy structures was calculated by distance geometry followed by an iterative relaxation-matrix-refinement procedure. The rmsd of the backbone coordinates with respect to the average structure was 17 pm. The two distinct thioether ring systems 1-6 and 7-19 were even better defined, with backbone rmsd of 10 pm and 14 pm, respectively. Actagardine shows a rigid compact globular shape based on the constraining bridging pattern, which is composed of an N-terminal lanthionine ring from residues 1-6 and three intertwined C-terminal methyllanthionine rings comprising residues 7-12, 9-17 and 14-19. In addition, this C-terminal ring system is stabilised by a short antiparallel beta sheet. A feature of the actagardine structure is the presence of two putative binding pockets. A pocket is generated by the covalent constraints of the C-terminal thioether ring system. The rim of this pocket is built up by a loop structure comprising residues 12-19, whose backbone amide protons are all directed to the centre of the pocket. The second pocket is formed by an L-shaped orientation of the N-terminal and C-terminal thioether ring systems. The only two hydrophilic amino acid residues of actagardine, Glu11 and Ser2, are directed to this pocket. A region of high sequence similarity with the related lantibiotic mersacidin is located exactly at the position of the second pocket (residues 3-12). This suggests that the second pocket is responsible for the antibiotic mode of action of actagardine and mersacidin as inhibitors of the murein biosynthesis of gram-positive bacteria.

The primary structure of the peptide lantibiotic actagardine was determined in (2H3)acetonitrile/H2O by homonuclear two- and three-dimensional NMR spectroscopy as well as 2D 1H(13C) correlation spectra at natural abundance. Actagardine was found to be a tetracyclic 19-residue peptide containing one lanthionine and three overlapping beta-methyllanthionine bridges. Sequential resonance assignment and location of the four thioether rings was accomplished by 2D NOESY, 3D NOESY-TOCSY and gradient-enhanced 1H(13C)-HMBC spectra. The C-terminal thioether bridge was shown to be oxidized to a sulfoxide. The NMR data were additionally confirmed by mass spectrometry and Edman degradation after chemical modification, which allowed sequencing of lanthionine and beta-methyllanthionine residues. Our studies clearly show, that the structure of actagardine as previously published by Kettenring et al. (1990) J. Antibiot. 43, 1082-1088 is not correct.