Heptapeptide microcystin and pentapeptide motuporin (nodularin-V) are equipotent inhibitors of type-1 and type-2A protein phosphatase catalytic subunits (PP-1c and PP-2Ac). Herein we describe elucidation of the molecular mechanisms involved in the interaction of these structurally similar hepatotoxins with PP-1c/PP-2Ac and identification of an important functional difference between their mode of interaction with these enzymes. Microcystin-LR, microcystin-LA, and microcystin-LL were found to interact with PP-2Ac and PP-1c by a two-step mechanism involving rapid binding and inactivation of the protein phosphatase (PPase) catalytic subunit, followed by a slower covalent interaction (within hours). Covalent adducts comprising PPase-toxin complexes were separated from free PPase by C-18 reverse-phase liquid chromatography, thus allowing the time course of covalent adduct formation to be quantitated. In contrast to microcystins, motuporin (nodularin-V) and nodularin-R were unable to form covalent complexes with either PP-1c or PP-2Ac even after 96 h incubation. Specific reduction of microcystin-LA to dihydromicrocystin-LA abolished the ability of the toxin to form a covalent adduct with PP-2Ac. Specific methyl esterification of the single Glu residue in microcystin-LR rendered this toxin inactive as a PPase inhibitor and abolished subsequent formation of a covalent adduct. Our data indicate that inactivation of PP-2Ac/PP-1c by microcystins precedes covalent modification of the PPases via a Michael addition reaction between a nucleophilic phosphatase residue and Mdha in the heptapeptide toxin. In contrast, following rapid inactivation of PP-2Ac/PP-1c by motuporin, the equivalent N-methyldehydrobutyrine residue in this toxin is unreactive and does not form a covalent bond with the PPases. These results are consistent with structural data for (i) the NMR solution structures of microcystin-LR and motuporin, which indicate a striking difference in the relative positions of their corresponding dehydroamino acids in the toxin peptide backbone, and (ii) X-ray crystallographic data on an inactive complex between PP-1c and microcystin-LR, which show a covalent bond between Cys-273 and the bound toxin.

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The chemically unique nature of the C20 beta-amino acid (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6- dienoic acid (Adda) portion of the microcystins has been exploited to develop a strategy to analyze for the total microcystin-LR (1; see Figure 1) burden in salmon liver and crab larvae tissues. Lemieux oxidation of microcystin-LR (1) gives 2-methyl-3-methoxy-4-phenylbutanoic acid (2), a unique marker for the presence of microcystins. The butanoic acid 2 can be isolated and detected by GC/MS from the livers of Atlantic salmon that received an ip injection of microcystin-LR (1) and from tissues of wild-caught crab larvae. The Lemieux oxidation-GC/MS results are compared with those from MeOH extraction-PPase analysis. Only approximately 24% of the total microcystin-LR (1) burden in salmon liver tissue is found to be extractable with MeOH. Similarly, the Lemieux oxidation-GC/MS method detected 10,000-fold greater microcystin concentrations in Cypress Island Dungeness crab larvae than did the MeOH extraction-PPase method. The disparity in microcystin concentrations measured by the two methods is taken as direct evidence for the existence of covalently bound microcystins in vivo.