EVIDENCE THAT MICROCYSTIN IS A THIO‐TEMPLATE PRODUCT¹

Abstract

The hepatotoxic cyclic heptapeptide toxins of cyanobacteria, collectively termed microcystins, are potent inhibitors of protein phosphatases PP1 and PP2A. The structure of microcystins resembles small, cyclic peptide secondary metabolites from fungi and eubacteria. Many of these metabolites are manufactured via a nonribosomal thio‐template mechanism. We submit evidence that microcystin is synthesized by a similar mechanism. The organism used in this study was Microcystis aeruginosa PCC7820. Using the traditional ATP‐32PPi exchange assay for thio‐template activity, we found activity in the presence of the substrate d‐amino acids occurring in microcystin. Thio‐template mechanisms are known to be unaffected by protein synthesis inhibitors such as chloramphenicol. We subjected cultures in exponential and stationary growth to chloramphenicol and monitored culture health versus toxicity. Although the health of the treated cultures declined, the toxicity of the remaining cells increased. We developed an in vitro assay to measure microcystin synthesis in cell lysates in the presence of chloramphenicol. By supplementing the lysates with ATP and the substrate amino acids present in microcystin, we detected a fourfold increase in total microcystins over the course of 20 min.

Mechanism study on the frequent variations of cell-bound microcystins in cyanobacterial blooms in Lake Taihu: Implications for water quality monitoring and assessments

“…However, in the following 20 years, very few studies have been carried out to confirm these findings. It has been reported that microcystin was synthesized in vivo by mcy genes in some cyanobacterial genera, including Microcystis and Anabaena (Arment and Carmichael, 1996;Dittman et al, 1997), and mcyB gene was commonly used as an important indicator of toxin-producing ability for toxic cyanobacteria (Kurmayer et al, 2003). For example, Kurmayer and his colleagues determined the presence of mcy genes in different colonies of Microcystis blooms from a number of freshwater lakes, and found the mcyB gene in most of the M. aeruginosa colonies (>73%) but only in <16% of the M. ichthyoblabe colonies, whereas not in Microcystis wesenbergii colonies (Kurmayer et al, 2003).…”

Natural Variation in the Microcystin Synthetase Operon mcyABC and Impact on Microcystin Production in Microcystis Strains

“…Microcystin is produced nonribosomally by the microcystin synthetase enzyme complex via a thio-template mechanism (2). Nonribosomal peptide synthetase genes consist of modules that are built up of domains, and each module activates one amino acid, which is incorporated into the growing peptide chain in the order in which the modules are arranged.…”

Multiple Alternate Transcripts Direct the Biosynthesis of Microcystin, a Cyanobacterial

“…Microcystin is a hepatotoxic heptapeptide produced by several species of the genera Microcystis, Anabaena, Nostoc, and Oscillatoria. More than 65 structural isoforms of microcystins with various toxicities have been identified having the common structure cyclo(Adda-D-Glu-Mdha-D-Ala-L-X-D-MeAsp-L-Z), where X and Z are variable L amino acids, Adda is 3-amino-9-methoxy-2,6,8,-trimethyl-10-phenyl-4,6-decadienoic acid, D-MeAsp is 3-methylaspartic acid, and Mdha is N-methyldehydroalanine (27, 31).Microcystin is synthesized nonribosomally via a mixed polyketide synthase/nonribosomal peptide synthetase system called microcystin synthetase (2,12,23). Recently, the gene cluster encoding the microcystin synthetase complex has been identified and sequenced (25,34).…”

“…Microcystin is synthesized nonribosomally via a mixed polyketide synthase/nonribosomal peptide synthetase system called microcystin synthetase (2,12,23). Recently, the gene cluster encoding the microcystin synthetase complex has been identified and sequenced (25,34).…”