Saxitoxin (STX) and its 57 analogs are a broad group of natural neurotoxic alkaloids, commonly known as the paralytic shellfish toxins (PSTs). PSTs are the causative agents of paralytic shellfish poisoning (PSP) and are mostly associated with marine dinoflagellates (eukaryotes) and freshwater cyanobacteria (prokaryotes), which form extensive blooms around the world. PST producing dinoflagellates belong to the genera Alexandrium, Gymnodinium and Pyrodinium whilst production has been identified in several cyanobacterial genera including Anabaena, Cylindrospermopsis, Aphanizomenon Planktothrix and Lyngbya. STX and its analogs can be structurally classified into several classes such as non-sulfated, mono-sulfated, di-sulfated, decarbamoylated and the recently discovered hydrophobic analogs—each with varying levels of toxicity. Biotransformation of the PSTs into other PST analogs has been identified within marine invertebrates, humans and bacteria. An improved understanding of PST transformation into less toxic analogs and degradation, both chemically or enzymatically, will be important for the development of methods for the detoxification of contaminated water supplies and of shellfish destined for consumption. Some PSTs also have demonstrated pharmaceutical potential as a long-term anesthetic in the treatment of anal fissures and for chronic tension-type headache. The recent elucidation of the saxitoxin biosynthetic gene cluster in cyanobacteria and the identification of new PST analogs will present opportunities to further explore the pharmaceutical potential of these intriguing alkaloids.

The cyanobacteria or “blue-green algae”, as they are commonly termed, comprise a diverse group of oxygenic photosynthetic bacteria that inhabit a wide range of aquatic and terrestrial environments, and display incredible morphological diversity. Many aquatic, bloom-forming species of cyanobacteria are capable of producing biologically active secondary metabolites, which are highly toxic to humans and other animals. From a toxicological viewpoint, the cyanotoxins span four major classes: the neurotoxins, hepatotoxins, cytotoxins, and dermatoxins (irritant toxins). However, structurally they are quite diverse. Over the past decade, the biosynthesis pathways of the four major cyanotoxins: microcystin, nodularin, saxitoxin and cylindrospermopsin, have been genetically and biochemically elucidated. This review provides an overview of these biosynthesis pathways and additionally summarizes the chemistry and toxicology of these remarkable secondary metabolites.

Nodularia spumigena is a bloom-forming cyanobacterium which produces the hepatotoxin nodularin. The complete gene cluster encoding the enzymatic machinery required for the biosynthesis of nodularin in N. spumigena strain NSOR10 was sequenced and characterized. The 48-kb gene cluster consists of nine open reading frames (ORFs), ndaA to ndaI, which are transcribed from a bidirectional regulatory promoter region and encode nonribosomal peptide synthetase modules, polyketide synthase modules, and tailoring enzymes. The ORFs flanking the nda gene cluster in the genome of N. spumigena strain NSOR10 were identified, and one of them was found to encode a protein with homology to previously characterized transposases. Putative transposases are also associated with the structurally related microcystin synthetase (mcy) gene clusters derived from three cyanobacterial strains, indicating a possible mechanism for the distribution of these biosynthetic gene clusters between various cyanobacterial genera. We propose an alternative hypothesis for hepatotoxin evolution in cyanobacteria based on the results of comparative and phylogenetic analyses of the nda and mcy gene clusters. These analyses suggested that nodularin synthetase evolved from a microcystin synthetase progenitor. The identification of the nodularin biosynthetic gene cluster and evolution of hepatotoxicity in cyanobacteria reported in this study may be valuable for future studies on toxic cyanobacterial bloom formation. In addition, an appreciation of the natural evolution of nonribosomal biosynthetic pathways will be vital for future combinatorial engineering and rational design of novel metabolites and pharmaceuticals.

Summary Over the past 15 years, the genetic basis for production of many cyanobacterial bioactive compounds has been described. This knowledge has enabled investigations into the environmental factors that regulate the production of these toxins at the molecular level. Such molecular or systems level studies are also likely to reveal the physiological role of the toxin and contribute to effective water resource management. This review focuses on the environmental regulation of some of the most relevant cyanotoxins, namely the microcystins, nodularin, cylindrospermopsin, saxitoxins, anatoxins and jamaicamides.

Aromatic amino acid ammonia-lyases catalyze the deamination of L-His, L-Phe, and L-Tyr, yielding ammonia plus aryl acids bearing an alpha,beta-unsaturated propenoic acid. We report crystallographic analyses of unliganded Rhodobacter sphaeroides tyrosine ammonia-lyase (RsTAL) and RsTAL bound to p-coumarate and caffeate. His 89 of RsTAL forms a hydrogen bond with the p-hydroxyl moieties of coumarate and caffeate. His 89 is conserved in TALs but replaced in phenylalanine ammonia-lyases (PALs) and histidine ammonia-lyases (HALs). Substitution of His 89 by Phe, a characteristic residue of PALs, yields a mutant with a switch in kinetic preference from L-Tyr to L-Phe. Structures of the H89F mutant in complex with the PAL product, cinnamate, or the PAL-specific inhibitor, 2-aminoindan-2-phosphonate (AIP), support the role of position 89 as a specificity determinant in the family of aromatic amino acid ammonia-lyases and aminomutases responsible for beta-amino acid biosynthesis.

Phenylalanine ammonia lyase (PAL)1 catalyzes the deamination of phenylalanine to cinnamate and ammonia. While PALs are common in terrestrial plants where they catalyze the first committed step in the formation of phenylpropanoids, only a few prokaryotic PALs have been identified to date. Here we describe for the first time PALs from cyanobacteria, in particular Anabaena variabilis ATCC 29413 and Nostoc punctiforme ATCC 29133, identified by screening the genome sequences of these organisms for members of the aromatic amino acid ammonia lyase family. Both PAL genes associate with secondary metabolite biosynthetic gene clusters as observed for other eubacterial PAL genes. In comparison to eukaryotic homologues, the cyanobacterial PALs are 20% smaller in size, but share similar substrate selectivity and kinetic activity toward L-phenylalanine over L-tyrosine. Structure elucidation by protein x-ray crystallography confirmed that the two cyanobacterial PALs are similar in tertiary and quatenary structure to plant and yeast PALs as well as the mechanistically related histidine ammonia-lyases.

Structural inspection of the bacterial meroterpenoid antibiotics belonging to the napyradiomycin family of chlorinated dihydroquinones suggests that the biosynthetic cyclization of their terpenoid subunits is initiated via a chloronium ion. The vanadium-dependent haloperoxidases that catalyze such reactions are distributed in fungi and marine algae and have yet to be characterized from bacteria. The cloning and sequence analysis of the 43-kb napyradiomycin biosynthetic cluster (nap) from Streptomyces aculeolatus NRRL 18422 and from the undescribed marine sediment-derived Streptomyces sp. CNQ-525 revealed 33 open reading frames, three of which putatively encode vanadium-dependent chloroperoxidases. Heterologous expression of the CNQ-525-based nap biosynthetic cluster in Streptomyces albus produced at least seven napyradiomycins, including the new analog 2-deschloro-2-hydroxy-A80915C. These data not only revealed the molecular basis behind the biosynthesis of these novel meroterpenoid natural products but also resulted in the first in vivo verification of vanadium-dependent haloperoxidases.Nature has devised several mechanisms to polarize the terminal olefin of linear terpenes to facilitate the creation of new C-X bonds. For instance, cyclization of the C 30 hydrocarbon squalene to steroids and hopanoids is initiated, respectively, by epoxidation or protonation of the terminal olefin. Although these biosynthetic strategies are widely distributed, a third mechanism for terpene cyclization has been characterized in marine macroalgae involving bromonium ion-induced ring closure (1-3). Oxidation of the halide is catalyzed by vanadiumdependent bromoperoxidase in the presence of hydrogen peroxide to produce the corresponding hypohalous acid. This species then further reacts with electron-rich organic substrates in a regio-and stereoselective manner, giving rise to brominated terpenes and other halogenated natural products (1, 2, 4). Vanadium-dependent bromoperoxidases are widely distributed in marine algae, and the first enzyme was discovered in 1984 from the brown alga Ascophyllum nodosum (5).Vanadium chloroperoxidases (V-ClPOs), 3 on the other hand, have been isolated primarily from dematiaceous hyphomycete fungi (1). The first enzyme was characterized in 1993 from Curvularia inaequalis (6), and even though there are numerous chlorinated marine natural products, V-ClPOs have not been reported from marine organisms to date (1, 2, 7). Although the biological function of V-ClPOs has not yet been elucidated, marine algal vanadium-dependent bromoperoxidases have been shown through in vitro chemoenzymatic conversions to catalyze bromonium ion-initiated cyclization of terpenes and ethers (1,8). These studies not only demonstrated that the enzymes were able to initiate cyclization of a terpene by a bromonium ion but also proved that the halogenation reaction occurred with stereochemical control. To date, all known V-dependent haloperoxidases have been characterized in vitro from eukaryotic systems (9).Structural inspection of...

BackgroundThe hapalindole-type family of natural products is a group of hybrid isoprenoid-indole alkaloids, produced solely by members of the Subsection V cyanobacterial strains. This family broadly includes the hapalindoles, welwitindolinones, fisherindoles and ambiguines amongst others, all of which have an isonitrile- or isothiocyanate-containing indole alkaloid skeleton, with a cyclized isoprene unit. The hapalindoles are diversified into the welwitindolinones, fischerindoles and ambiguines through the employment of tailoring oxygenase, methyltransferase and prenyltransferase enzymes. We compare the genetic basis for the biosynthesis of this diverse group of natural products and identify key early biosynthetic intermediates.ResultsWhole genome sequencing of freshwater and terrestrial cyanobacteria Westiella intricata UH strain HT-29-1, Hapalosiphon welwitschii UH strain IC-52-3, Fischerella ambigua UTEX 1903 and Fischerella sp. ATCC 43239 led to the identification of a candidate hapalindole-type gene cluster in each strain. These were compared with the recently published ambiguine and welwitindolinone gene clusters and four unpublished clusters identified within publicly available genomes. We present detailed comparative bioinformatic analysis of the gene clusters and the biosynthesis of a pivotal indole-isonitrile intermediate resulting in both cis and trans geometrical isomers. Enzyme analyses and metabolite extractions from two hapalindole-producing Fischerella strains indicate the presence of cis and trans indole-isonitriles as biosynthetic intermediates in the early steps of the pathway.ConclusionsInterestingly, the organization of the welwitindolinone gene cluster is conserved in all producing strains but distinct from the hapalindole and ambiguine clusters. Enzymatic assays using WelI1 and WelI3 from Westiella intricata UH strain HT-29-1 demonstrated the ability to catalyze the formation of both cis and trans geometrical isomers when using a cell lysate. The enzymatic and metabolic characterization of both cis and trans indole-isonitrile intermediates implies conservation of their stereochemical integrity towards members of the ambiguine and welwitindolinone products. In summary, we present data that supports a unified biosynthetic pathway towards hapalindoles in nine individual species of cyanobacteria. Diversification of the pathway occurs later through the employment of specialized enzymatic steps towards fischerindoles, ambiguines and welwitindolinones.