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Subcellular Localization of Dinoflagellate Polyketide Synthases and Fatty Acid Synthase Activity1

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Subcellular Localization of Dinoflagellate Polyketide Synthases and Fatty Acid Synthase Activity1 J. Phycol. 49, 1118–1127 (2013) © Published 2013. This article is a U.S. Government work and is in the public domain in the U.S.A. DOI: 10.1111/jpy.12120 SUBCELLULAR LOCALIZATION OF DINOFLAGELLATE POLYKETIDE SYNTHASES AND FATTY ACID SYNTHASE ACTIVITY1 Frances M. Van Dolah,2 Mackenzie L. Zippay Marine Biotoxins Program, NOAA Center for Coastal Environmental Health and Biomolecular Research, Charleston, South Carolina 29412, USA Marine Biomedical and Environmental Sciences, Medical University of South Carolina, Charleston, South Carolina 29412, USA Laura Pezzolesi Interdepartmental Research Centre for Environmental Science (CIRSA), University of Bologna, Ravenna 48123, Italy Kathleen S. Rein Department of Chemistry and Biochemistry, Florida International University, Miami, Florida 33199, USA Jillian G. Johnson Marine Biotoxins Program, NOAA Center for Coastal Environmental Health and Biomolecular Research, Charleston, South Carolina 29412, USA Marine Biomedical and Environmental Sciences, Medical University of South Carolina, Charleston, South Carolina 29412, USA Jeanine S. Morey, Zhihong Wang Marine Biotoxins Program, NOAA Center for Coastal Environmental Health and Biomolecular Research, Charleston, South Carolina 29412, USA and Rossella Pistocchi Interdepartmental Research Centre for Environmental Science (CIRSA), University of Bologna, Ravenna 48123, Italy Dinoflagellates are prolific producers of polyketide related to fatty acid synthases (FAS), we sought to secondary metabolites. Dinoflagellate polyketide determine if fatty acid biosynthesis colocalizes with synthases (PKSs) have sequence similarity to Type I either chloroplast or cytosolic PKSs. [3H]acetate PKSs, megasynthases that encode all catalytic domains labeling showed fatty acids are synthesized in the on a single polypeptide. However, in dinoflagellate cytosol, with little incorporation in chloroplasts, PKSs identified to date, each catalytic domain resides consistent with a Type I FAS system. However, on a separate transcript, suggesting multiprotein although 29 sequences in a K. brevis expressed sequence tag database have similarity (BLASTx e- complexes similar to Type II PKSs. Here, we provide À evidence through coimmunoprecipitation that value <10 10) to PKSs, no transcripts for either Type I single-domain ketosynthase and ketoreductase (cytosolic) or Type II (chloroplast) FAS are present. proteins interact, suggesting a predicted multiprotein Further characterization of the FAS complexes may complex. In Karenia brevis (C.C. Davis) Gert Hansen help to elucidate the functions of the PKS enzymes & Ø. Moestrup, previously observed chloroplast identified in dinoflagellates. localization of PKSs suggested that brevetoxin Key index words: brevetoxin; Coolia monotis; dinofla- biosynthesis may take place in the chloroplast. Here, gellates; fatty acid synthase; Karenia brevis; Ostreopsis we report that PKSs are present in both cytosol and cf. ovata; palytoxin; polyketide synthase chloroplast. Furthermore, brevetoxin is not present in isolated chloroplasts, raising the question of what List of abbreviations: ACP, acyl carrier protein; chloroplast-localized PKS enzymes might be doing. AT, acyl transferase; CHAPS, 3[(3-cholamidopropyl) Antibodies to K. brevis PKSs recognize cytosolic and dimethylammonio]-propanesulfonic acid; CIB, chloroplast proteins in Ostreopsis cf. ovata Fukuyo, chloroplast isolation buffer; DAPI, 4′,6-diamidin- and Coolia monotis Meunier, which produce different 2-phenylindole; DH, dehydratase; DPM, distintegra- suites of polyketide toxins, suggesting that these PKSs tions per minute; EDTA, Ethylenediaminetetraacetic may share common pathways. Since PKSs are closely acid; EST, expressed sequence tag; FAME, fatty acid methyl ester; FAS, fatty acid synthase; IP, 1 immunoprecipitation; KR, ketoreductase; KS, ketoa- Received 22 May 2013. Accepted 25 August 2013. – 2Author for correspondence: e-mail [email protected]. cyl synthase; LC-MS, liquid chromatography mass Editorial Responsibility: C. Bowler (Associate Editor) spectrometry; LOD, limit of detection; MRM, multi- 1118 DINOFLAGELLATE POLYKETIDE SYNTHASES 1119 ple reaction monitoring; PBS, phosphate-buffered tain multiple modules on a single polypeptide, each saline; PKS, polyketide synthase; SIM, selected ion of which contains all active site domains needed to monitoring; TBS, tris-buffered saline; TBST, tris- carry out one chain extension, where each module buffered saline with tween; TE, thioesterase used only once during polyketide assembly. In con- trast, bacterial Type II PKSs are organized as com- plexes of smaller proteins where each catalytic Marine dinoflagellates produce some of the most domain is located on a separate peptide, a structure potent toxins on earth, and are responsible for similar to Type II FAS present in bacteria and chlo- more than 60,000 intoxication incidents per year on roplasts of higher plants and some algae. Type III a worldwide basis (Van Dolah 2000). The majority PKSs are small, structurally divergent proteins of dinoflagellate toxins that adversely affect human typified by plant chalcone synthases, and melanin health are polyether compounds, synthesized by producing PKSs in bacteria (Hopwood 1997, Funa complex enzymes known as PKS (Shimizu 2003). et al. 1999, Khosla et al. 1999, Gross et al. 2006). Polyketides are synthesized in a manner analogous PKSs have a patchy distribution among protist lin- to fatty acid biosynthesis through the sequential eages for which genomic sequence data are available addition of carboxylic acid building blocks: the con- (John et al. 2008): type I PKSs are present in chlo- densation reaction between carboxylic acids is per- rophytes (Chlamydomonas reinhardtii and Ostreococcus formed by a b-KS domain, whereupon the b-keto spp.) and the haptophyte Emiliania huxleyi, but are group may be fully or partially reduced by successive absent from stramenopiles (Thallassiosira pseudonan- activities of KR, DH, and enoyl reductase domains na, Phaeodactylum tricornutim, Phytophthora spp.) and following each chain elongation step (Staunton and Excavata (Trypanosoma spp., Leishmania major, Naegle- Weissman 2001). The growing carbon chain resides ria gruberi, Monosiga brevicollis). Transcriptome on a phosphopantetheine “arm” on the ACP that sequencing identified Type I PKSs in Prymnesio- “swings” it into close proximity of the catalytic sites, phytes, Chrysochromulina polylepis (John et al. 2010), while an AT brings additional “extender” carboxylic and Prymnesium parvum (Freitag et al. 2011). Among acid units to be added to the growing chain. The the closest relatives to dinoflagellates, Type I PKSs full-length polyketide is then released from the PKS are absent from the ciliate, Tetrahymena thermophile, complex by a TE, and post-PKS modifications create but are variably present in the Apicomplexa (pres- the final polyketide structure. ent in Cryptosporidium spp., Toxoplasma gondii, and PKSs are structurally and functionally similar to Eimeria tenella, but absent from Plasmodium FASs and likely evolved from an ancestral condens- falciparum and Theileria parva). The Type I PKS in ing enzyme that served to make cellular functions Cryptosporidium parvum is a 40 kb, intronless gene more efficient (Hopwood 1997). The addition of an that encodes for a single 13,000 amino acid ACP and AT produced a primitive PKS, while fur- modular protein (Zhu et al. 2002). ther additions of a reductive cycle converted the Because of the large, complex genomes of dino- primitive PKS to an FAS. The fully reductive FAS flagellates (e.g. 1 9 1011 bp in K. brevis), the avail- became fixed in primary metabolism as fatty acids ability of genomic sequences has been limited to became essential components of the cell. PKSs sub- date. In the amphidinolide producer Amphidinium,a sequently diverged, retaining variable numbers of predicted 5,625 bp PKS was found, containing sev- reductive catalytic domains to produce a variety of eral PKS domains (KS, AT, DH, KR, ACP, and TE; endproducts. Recent studies in plants suggest that Kubota et al. 2006). However, many unexplained the duplication of genes involved in primary metab- frame shifts and gaps were present in the sequence. olism is the primary source of new genetic material Similarly, Bachvaroff and Place (2008) found for secondary metabolism. Following duplication, numerous large introns within a genomic sequence even minor sequence changes in one copy can alter for a PKS KR domain protein in Amphidinium carte- substrate specificity, leading to new secondary rae that far exceeds the size of its coding region. metabolism products (Ober 2005). Since secondary Degenerate PCR identified partial sequences con- metabolites are not essential for survival, these taining type I PKS KS domains in seven dinoflagel- genes are more tolerant of mutations than their late species, including two encoded by K. brevis counterparts in primary metabolism, further pro- (Snyder et al. 2003, 2005). Using a cDNA library moting the diversity observed in secondary metabo- screening approach, Monroe and Van Dolah (2008) lites (Weng et al. 2012). identified K. brevis transcripts with sequence similar- PKSs are typically categorized as Types I–III, ity to type I PKSs. However, each transcript con- although these divisions are becoming less clear as tained only a single catalytic domain, a structure more PKSs are characterized. Type I are large pro- more similar to type II PKSs. Homologous single- teins with multiple active sites on a single polypep- domain Type I-like PKS
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