Conformations of Cyanobacterial Hepatotoxins Microcystin-LR and Nodularin

Arto Annila 1), Maarit Kontteli 1), Jaana Lehtimäki 2), Kaarina Sivonen 2), Jussi Meriluoto 3) and John Eriksson 3)

1) VTT Chemical Technology, POB 1401, FIN-02044 VTT, Finland

2) Department of Applied Chemistry and Microbiology, POB 27, Viikki, FIN-00014 University of Helsinki, Finland

3) Turku Centre for Biotechnology POB 123, BioCity, FIN-20521 Turku, Finland

Microcystin-LR from Microcystis aeruginosa and nodularin from Nodularia spumigena, are extremely hepatotoxic and also potent tumor promoters. These cyanobacterial toxins inhibit the type 1 and type 2A serine/threonine-specific protein phosphatases, essential enzymes that regulate cell growth and differentiation.

We have determined the three-dimensional structures of microcystin-LR nodularin in water by nuclear magnetic resonance spectroscopy. Proton resonances were assigned from two-dimensional spectra. Inter proton distances as well as dihedral angles were extracted for structure calculations. The conformational space was sampled by distance geometry. Only one family of structures were found for each molecule.

The cyclic backbone for both molecules adopts a saddle-shaped conformation. The backbone ring of nodularin is less buckled than that of microcystin-LR. The root mean square deviation for the heavy atoms in the backbone was approximately 0.5 Å whereas the long side chains of Adda and Arg appear flexible because their coordinates were not obtained as precisely. Variable temperature experiments indicate that NH of MAsp, Adda, IGlu and Leu in microcystin-LR are not readily accessible to solvent. A similar result was obtained from hydrogen to deuterium exchange measurements. Thus there are intra molecular hydrogen bonds both in microcystin-LR and nodularin. The homologous region MAsp-Arg-Adda-IGlu has a very similar spatial arrangement in both toxins. It seems that this region contains the epitope for tight binding since there are earlier indications that parts of MAsp, Adda and IGlu account for the protein phosphatase binding and consequently for the poisonous properties.