microcystin and dehydroalanine

Microcystins form a large family of small cyclic heptapeptides harbouring extensive modifications in amino acid residue composition and functional group chemistry. These peptide hepatotoxins contain a range of non-proteinogenic amino acids and unusual peptide bonds, and are typically N-methylated. They are synthesized on large enzyme complexes consisting of non-ribosomal peptide synthetases and polyketide synthases in a variety of distantly related cyanobacterial genera. Here we report a 1236 bp in-frame deletion mutation in the mcyA gene of the microcystin biosynthetic pathway in nine strains of the genus Anabaena. The deletion removed almost the entire N-methyltransferase (NMT) domain. Strains of Anabaena carrying the in-frame deletion mutation incorporated mainly dehydroalanine (Dha) into the microcystins they produce while strains with full-length mcyA genes incorporated mainly N-methyldehydroalanine (Mdha). Interestingly, the strains of Anabaena lacking the NMT domain also incorporated elevated amounts of L-Ser, the precursor of Mdha and Dha, into the microcystin they produced relative to strains carrying functional NMT domains. We provide evidence for the in-frame deletion of the NMT domain without the co-conversion of the flanking adenylation domain. Our results demonstrate a further example of the strategies employed by cyanobacteria in the biosynthesis of microcystin variants.

Topics: Alanine; Anabaena; Bacterial Proteins; DNA, Bacterial; Microcystins; Molecular Sequence Data; Molecular Structure; Peptide Synthases; Phylogeny; Polymerase Chain Reaction; Protein Structure, Tertiary; Sequence Analysis, DNA; Sequence Deletion; Sequence Homology; Serine; Water Microbiology

The interaction between protein phosphatase 1 (PP1) and microcystin (MC) was stable in 1% SDS or 70% formic acid indicative of a covalent interaction. Here we isolate the MC-binding peptide and demonstrate that Cys273 of PP1 binds covalently to the methyl-dehydroalanine (Mdha) residue of the toxin. Mutation of Cys273 to Ala, Ser or Leu abolished covalent binding to MC, as did reduction of the Mdha residue of the toxin with ethanethiol. The abolition of covalent binding increased the IC50 for toxin inhibition of PP1 by 5- to 20-fold. The covalent binding of MC to protein serine/threonine phosphatases explains the failure to detect this toxin post-mortem in suspected cases of MC poisoning.

Topics: Alanine; Amino Acid Sequence; Bacterial Toxins; Base Sequence; Cyanobacteria; Cysteine; Humans; Iodine Radioisotopes; Isotope Labeling; Microcystins; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptides; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Binding; Protein Phosphatase 1