Evidence for differences in the metabolism of saxitoxin and C1+2 toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii T3 Francesco Pomati a,b , Michelle C. Moffitt c , Rosalia Cavaliere b , Brett A. Neilan b, * a Environmental Research Group, DBSF University of Insubria, 21100 Varese, Italy b School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Govett Street, Sydney 2052, NSW, Australia c Division of Medicinal Chemistry, College of Pharmacy, University of Arizona, Tucson, AZ 85721-0207, USA Received 13 February 2004; received in revised form 19 May 2004; accepted 24 May 2004 Available online 17 June 2004 Abstract The activity of paralytic shellfish poisoning (PSP) toxins biosynthetic enzymes was assayed in the cyanobacterium Cylindrospermopsis raciborskii T3 after inhibiting protein synthesis with chloramphenicol (CAM). The production of C1 + 2 and saxitoxin (STX) was sensitive to CAM with STX levels decreasing by 70% after 24-h exposure to the antibiotic. PSP toxin production was strongly promoted by arginine supplementation, with a maximum 476% increase in intracellular STX concentrations after 24-h exposure to 10 mM of the amino acid. However, arginine had no stimulating effect on PSP toxin levels if supplemented in combination with CAM at 10 Agl  1 . Addition of agmatine and proline to C. raciborskii T3 cultures in the presence of 10 Agl  1 CAM increased C1 + 2 toxins levels, while having a negative or no effect on STX accumulation. In vitro, PSP toxin levels increased naturally in cyanobacterial extracts, with CAM and arginine having no influence on either C1 + 2 or STX synthesis. The evidence presented in this study suggests a possible difference between the metabolism of STX and the C1 + 2 toxins and indicated a high turnover rate of STX biosynthetic enzymes in C. raciborskii T3. D 2004 Elsevier B.V. All rights reserved. Keywords: Arginine; Chloramphenicol; Cylindrospermopsis raciborskii; Cyanobacteria; C1+2 toxin; Saxitoxin 1. Introduction The paralytic shellfish poisoning (PSP) toxins are well- known natural bioactive compounds due to their acciden- tal consumption in contaminated seafood [1]. These mol- ecules, of which the most potent representative is saxitoxin (STX), are a class of neurotoxic alkaloids having different isoforms and varied toxicities [2]. PSP toxins can be either non-sulfated (STX, neosaxitoxin- neoSTX) or sulfated (gonyautoxins-GTX, C-toxins), and selectively block voltage-gated Na + channels in excitable cells thereby affecting the impulse generation in animals which can lead, in extreme cases, to death [1,3]. Saxitoxin and its analogue compounds have been reported to occur naturally in marine dinoflagellates [3–5], filamentous cyanobacteria [6–10], as well as certain heterotrophic bacteria [11]. The chemical structure and toxicity of PSP toxins have been described. In contrast, as reviewed by Cembella [12], little is known about the enzymatic pathway(s) leading to the biosynthesis of these peculiar alkaloids. Shimizu [13,14] demonstrated that arginine, acetate, and a methyl group (from S-adenosylmethionine) are incorporated into the tri- cyclic perhydropurine skeleton of STX in cyanobacteria and dinoflagellates (Fig. 1). Given this sequence of biochemical reactions, candidate enzymes in STX biosynthesis have been hypothesized [13,15]. In particular, the Claisen-type condensation between the a-carbon of arginine and C1 of acetate could be catalyzed by aminolevulinate synthase (involved in the metabolism of chlorophyll and heme synthesis). Alternatively, this reaction could involve a hybrid non-ribosomal peptide synthetases/polyketide syn- thases (NRPS/PKS) enzyme complex with an acyl adeny- lation, h-ketoacyl synthase and thioesterase domains. 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.05.006 Abbreviations: CAM, chloramphenicol; GTX, gonyautoxin; MCYST, microcystins; NRPS, non-ribosomal peptide synthetase; NODLN, nod- ularin; PKS, polyketide synthase; PSP, paralytic shellfish poisoning; STX, saxitoxin * Corresponding author. Tel.: +61-2-9385-3235; fax: +61-2-9385- 1591. E-mail address: b.neilan@unsw.edu.au (B.A. Neilan). www.bba-direct.com Biochimica et Biophysica Acta 1674 (2004) 60 – 67