Subcellular localization of dinoflagellate polyketide synthases and fatty acid synthase activity

Abstract

Dinoflagellates are prolific producers of polyketide secondary metabolites. Dinoflagellate polyketide synthases (PKSs) have sequence similarity to Type I PKSs, megasynthases that encode all catalytic domains on a single polypeptide. However, in dinoflagellate PKSs identified to date, each catalytic domain resides on a separate transcript, suggesting multiprotein complexes similar to Type II PKSs. Here, we provide evidence through coimmunoprecipitation that single-domain ketosynthase and ketoreductase proteins interact, suggesting a predicted multiprotein complex. In Karenia brevis (C.C. Davis) Gert Hansen & Ø. Moestrup, previously observed chloroplast localization of PKSs suggested that brevetoxin biosynthesis may take place in the chloroplast. Here, we report that PKSs are present in both cytosol and chloroplast. Furthermore, brevetoxin is not present in isolated chloroplasts, raising the question of what chloroplast-localized PKS enzymes might be doing. Antibodies to K. brevis PKSs recognize cytosolic and chloroplast proteins in Ostreopsis cf. ovata Fukuyo, and Coolia monotis Meunier, which produce different suites of polyketide toxins, suggesting that these PKSs may share common pathways. Since PKSs are closely related to fatty acid synthases (FAS), we sought to determine if fatty acid biosynthesis colocalizes with either chloroplast or cytosolic PKSs. [(3) H]acetate labeling showed fatty acids are synthesized in the cytosol, with little incorporation in chloroplasts, consistent with a Type I FAS system. However, although 29 sequences in a K. brevis expressed sequence tag database have similarity (BLASTx e-value <10(-10) ) to PKSs, no transcripts for either Type I (cytosolic) or Type II (chloroplast) FAS are present. Further characterization of the FAS complexes may help to elucidate the functions of the PKS enzymes identified in dinoflagellates.

Brevetoxin, the Dinoflagellate Neurotoxin, Localizes to Thylakoid Membranes and Interacts with the Light‐Harvesting Complex II (LHCII) of Photosystem II

“…Brevetoxins have also been demonstrated to aggregate into artificial lipid bilayers 29. Our findings contradict earlier localization studies: one which failed to identify brevetoxin in isolated chloroplasts by LC‐MS/MS,30 and a published abstract that reported that brevetoxins are located in secretory vesicles by using fluorescent anti‐brevetoxin antibodies 31. A follow‐up paper to the latter report has not yet appeared.…”

Transcriptomic investigation into polyketide toxin synthesis in Ostreopsis (Dinophyceae) species

“…brevis found the absence of Type II FAS, suggesting that K . brevis might possess a Type I FAS system or involve the role of PKS‐like domains for fatty acid synthesis (Van Dolah et al ., ). Kohli et al .…”

“…In a study performed by Van Dolah et al . (), antibodies developed for K . brevis PKSs were used to bind to similar proteins from a strain of O .…”

Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate

“…Thus, the common ancestor of apicomplexans and dinoflagellates must have had both forms. Although FAS in dinoflagellates is less well studied, these organisms can synthesize fatty acids de novo (39,40). Gene disruption of type II FAS in Plasmodium shows that this plastid pathway is essential during the liver stage and, in a human strain, the insect stage of the life cycle, implying that FAS might be a function mandating plastid retention (13,35,41).…”

“…S1A). Polyketide syntases (PKS) are a related family of proteins that are considered degenerate FASs in which one or more modules have been lost, and PKS are known to occur in dinoflagellates (40). Although two of the transcripts contain all modules expected of complete type I FAS proteins, it is difficult to reliably discern FAS from PKS without biochemical validation.…”