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Molecular Cloning, Modeling, and Site-Directed Mutagenesis of Type III Polyketide Synthase from Sargassum binderi (Phaeophyta)

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Abstract

Type III polyketide synthases (PKSs) produce an array of metabolites with diverse functions. In this study, we have cloned the complete reading frame encoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi, and characterized the activity of its recombinant protein biochemically. The deduced amino acid sequence of SbPKS is 414 residues in length, sharing a higher sequence similarity with bacterial PKSs (38% identity) than with plant PKSs. The Cys-His-Asn catalytic triad of PKS is conserved in SbPKS with differences in some of the residues lining the active and CoA binding sites. The wild-type SbPKS displayed broad starter substrate specificity to aliphatic long-chain acyl-CoAs (C6–C14) to produce tri- and tetraketide pyrones. Mutations at H331 and N364 caused complete loss of its activity, thus suggesting that these two residues are the catalytic residues for SbPKS as in other type III PKSs. Furthermore, H227G, H227G/L366V substitutions resulted in increased tetraketide-forming activity, while wild-type SbPKS produces triketide α-pyrone as a major product. On the other hand, mutant H227G/L366V/F93A/V95A demonstrated a dramatic decrease of tetraketide pyrone formation. These observations suggest that His227 and Leu366 play an important role for the polyketide elongation reaction in SbPKS. The conformational changes in protein structure especially the cavity of the active site may have more significant effect to the activity of SbPKS compared with changes in individual residues.

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References

  • Abe I, Sano Y, Takahashi Y, Noguchi H (2003) Site-directed mutagenesis of benzalacetone synthase. The role of Phe215 in plant type II polyketide synthases. J Biol Chem 278:25218–25226

    Article  PubMed  CAS  Google Scholar 

  • Abe I, Utsumi Y, Oguro S, Noguchi H (2004) The first plant type III polyketide synthase that catalyzes formation of aromatic heptaketide. FEBS 562:171–176

    Article  CAS  Google Scholar 

  • Abe I, Oguro S, Utsumi Y, Sano Y, Noguchi H (2005a) Engineered biosynthesis of plant polyketides: chain length control in octaketide-producing plant type III polyketide synthase. J Am Chem Soc 127:12709–12716

    Article  PubMed  CAS  Google Scholar 

  • Abe I, Utsumi Y, Oguro S, Morita H, Sano Y, Noguchi H (2005b) A plant type III polyketide synthase that produces pentaketide chromone. J Am Chem Soc 127:1362–1363

    Article  PubMed  CAS  Google Scholar 

  • Abe I, Morita H, Oguro S, Noma H, Wanibuchi K, Kawahara N, Goda Y, Noguchi H, Kohno T (2007) Structure-based engineering of a plant type III polyketide synthase: formation of an unnatural nonaketide naphthopyrone. J Am Chem Soc 129:5976–5980

    Article  PubMed  CAS  Google Scholar 

  • Arnold TM, Targett NC (2002) Marine tannins: the importance of a mechanistic framework for predicting ecological roles. J Chem Ecol 28:1919–1934

    Article  PubMed  CAS  Google Scholar 

  • Austin MB, Saito T, Bowman ME, Haydock S, Kato A, Moore BS, Kay RR, Noel JP (2006) Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase. Nat Chem Biol 2:494–502

    Article  PubMed  CAS  Google Scholar 

  • Ben Ali A, De Baere R, Van der Auwera G, De Wachter R, Van de Peer Y (2001) Phylogenetic relationships among algae based on complete large-subunit rRNA sequences. Int J Syst Evol Microbiol 51:737–749

    Article  PubMed  CAS  Google Scholar 

  • Benkert P, Tosatto SCE, Schomburg D (2008) QMEAN: a comprehensive scoring function for model quality assessment. Prot Struct Funct Bioinform 71:261–277

    Article  CAS  Google Scholar 

  • Brand S, Hölscher D, Schierhorn A, Svatoš A, Schröder J, Schneider B (2006) A type III polyketide synthase from Wachendorfia thyrsiflora and its role in diarylheptanoid and phenylphenalenone biosynthesis. Planta 224:413–428

    Article  PubMed  CAS  Google Scholar 

  • El Hattab M, Bouzidi N, Ortalo-Magné A, Daghbouche Y, Richou M, Chitour SE, de Reviers B, Piovetti L (2009) Eicosapentaenoic acid: possible precursor of the phloroglucinol derivatives isolated from the brown alga Zonaria tournefortii (J.V. Lamouroux) Montagne. Biochem Syst Ecol 37:55–58

    Article  CAS  Google Scholar 

  • Felsenstein J (1989) PHYLIP—phylogeny inference package (Version 3.2). Cladistics 5:164–166

    Google Scholar 

  • Ferrer JL, Jez JM, Bowman ME, Dixon RA, Noel JP (1999) Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. Nat Prod Rep 20:79–110

    Google Scholar 

  • Funa N, Ohnishi Y, Ebizuka Y, Horinouchi S (2002) Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis. Biochem J 367:781–789

    Article  PubMed  CAS  Google Scholar 

  • Glombitza KW, Keusgen M, Hauperich S (1997) Fucophlorethols from the brown algae Sargassum spinuligerum and Cystophora torulosa. Phytochemistry 46:1417–1422

    Article  CAS  Google Scholar 

  • Gross F, Luniak N, Perlova O, Gaitatzis N, Jenke-Kodama H, Gerth K, Gottschalk D, Dittmann E, Müller R (2006) Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads. Arch Microbiol 185:28–38

    Article  PubMed  CAS  Google Scholar 

  • Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723

    Article  PubMed  CAS  Google Scholar 

  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  • Hopwood DA, Sherman DH (1990) Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Genet 23:37–66

    Article  Google Scholar 

  • Jeong J-C, Srinivasan A, Grüschow S, Bach H, Sherman DH, Dordick JS (2005) Exploiting the reaction flexibility of a type III polyketide synthase through in vitro pathway manipulation. J Am Chem Soc 127:64–65

    Article  PubMed  CAS  Google Scholar 

  • Jez JM, Austin MB, Ferrer J-L, Bowman ME, Schröder J, Noel JP (2000) Structural control of polyketide formation in plant-specific polyketide synthases. Chem Biol 7:919–930

    Article  PubMed  CAS  Google Scholar 

  • Jez JM, Bowman ME, Noel JP (2001a) Structure-guided programming of polyketide chain-length determination in chalcone synthase. Biochemistry 40:14829–14838

    Article  PubMed  CAS  Google Scholar 

  • Jez JM, Ferrer J-L, Bowman ME, Austin MB, Schröder J, Dixon RA, Noel JP (2001b) Structure and mechanism of chalcone synthase-like polyketide synthases. J Ind Microbiol Biotech 27:393–398

    Article  CAS  Google Scholar 

  • Jez JM, Bowman ME, Noel JP (2002) Expanding the biosynthetic repertoire of plant type III polyketide synthases by altering starter molecule specificity. Proc Natl Acad Sci USA 99:5319–5324

    Article  PubMed  CAS  Google Scholar 

  • Keusgen M, Falk M, Walter JA, Glombitza KW (1997) A phloroglucinol derivative from the brown alga Sargassum spinuligerum. Phytochemistry 46:341–345

    Article  CAS  Google Scholar 

  • Khotimchenko SV (2008) fatty acid composition of seven Sargassum species. Phytochemistry 30:2639–2641

    Article  Google Scholar 

  • Laskowski RA, MacArthur MW, Moss D, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291

    Article  CAS  Google Scholar 

  • Lukačin R, Schreiner S, Silber K, Matern U (2005) Starter substrate specificities of wild-type and mutant polyketide synthases from Rutaceae. Phytochemistry 66:277–284

    Article  PubMed  Google Scholar 

  • Miyanaga A, Funa N, Awakawa T, Horinouchi S (2008) Direct transfer of starter substrate from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis. Proc Natl Acad Sci USA 105:871–876

    Article  PubMed  CAS  Google Scholar 

  • Mizuuchi Y, Shimokawa Y, Wanibuchi K, Noguchi H, Abe I (2008) Structure function analysis of novel type III polyketide synthases from Arabidopsis thaliana. Biol Pharm Bull 31:2205–2210

    Article  PubMed  CAS  Google Scholar 

  • Moore BS, Hopke JN (2001) Discovery of a new bacterial polyketide biosynthetic pathway. Chem Biochem 2:35–38

    CAS  Google Scholar 

  • Morita H, Kondo S, Oguro S, Noguchi H, Sugio S, Abe I, Kohno T (2007) Structural insight into chain-length control and product specificity of pentaketide chromone synthase from Aloe arborescens. Chem Biol 14:359–369

    Article  PubMed  CAS  Google Scholar 

  • Ōmura S, Ikeda H, Ishikawa J, Hanamoto A, Takahashi C, Shinose M, Takahashi Y, Horikawa H, Nakazawa H, Osonoe T, Kikuchi H, Shiba T, Sakaki Y, Hattori M (2001) Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. Proc Natl Acad Sci USA 98:12215–12220

    Article  PubMed  Google Scholar 

  • Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358

    PubMed  CAS  Google Scholar 

  • Pfeifer BA, Khosla C (2001) Biosynthesis of polyketides in heterologous hosts. Microbiol Mol Biol Rev 65:106–118

    Article  PubMed  CAS  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  • Sankaranarayanam R, Saxena P, Marathe UB, Gokhale RS, Shanmugam VM, Rukmini R (2004) A novel tunnel in mycobacterial type III polyketide synthase reveals the structural basis for generating diverse metabolites. Nat Struct Mol Biol 11:894–900

    Article  Google Scholar 

  • Saxena P, Yadav G, Mohanty D, Gokhale R (2003) A new family of type III polyketide synthases in mycobacterium tuberculosis. J Biol Chem 278:44780–44790

    Article  PubMed  CAS  Google Scholar 

  • Schröder J (1997) A family of plant-specific polyketide synthases: facts and predictions. Trends Plant Sci 2:373–378

    Article  Google Scholar 

  • Shen B (2000) Biosynthesis of aromatic polyketides. Top Curr Chem 109:1–51

    Article  Google Scholar 

  • Singh IP, Bharate SB (2006) Phloroglucinol compounds of natural origins. Nat Prod Rep 23:558–591

    Article  CAS  Google Scholar 

  • Stöckigt J, Zenk MH (1975) Chemical syntheses and properties of hydroxycinnamoyl-coenzyme a derivatives. Z Naturforsch 30c:352–358

    Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed  CAS  Google Scholar 

  • Wanibuchi K, Zhang P, Abe T, Morita H, Kohno T, Chen G, Noguchi H, Abe I (2007) An acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata. FEBS 274:1073–1082

    Article  CAS  Google Scholar 

  • Waterman PG, Mole S (1994) Analysis of plant metabolites. Methods in ecology series. Blackwell, Oxford

    Google Scholar 

  • Winkel-Shirley B (2002) Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol 5:218–223

    Article  PubMed  CAS  Google Scholar 

  • Wong TK-M, Ho C-L, Lee W-W, Rahim RA, Phang S-M (2007) Analyses of expressed sequence tags (ESTs) from Sargassum binderi (Phaeophyta). J Phycol 43:528–534

    Article  CAS  Google Scholar 

  • Zhang YM, Rao MS, Heath RJ, Price AC, Olson AJ, Rock CO, White SW (2001) J Biol Chem 276:8231–8238

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We would like to acknowledge the Universiti Putra Malaysia for the Graduate Research Fellowship awarded to Baharum H. This project was funded by eScienceFund grant number 02-01-04-SF0020 from the Ministry of Science, Technology and Innovation (MOSTI) of Malaysia (to Ho C.-L. and Ng K.-Y.), Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to I.A. and H.M.), and grants from Takeda Science Foundation (to H.M).

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Authors and Affiliations

  1. Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

    Hariyanti Baharum, Fong-Chin Lee, Raha Abdul Rahim & Chai-Ling Ho

  2. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bukyo-ku, Tokyo, 113-0033, Japan

    Hiroyuki Morita, Akifumi Tomitsuka & Ikuro Abe

  3. T-BioVenture Sdn Bhd, L6-03, Wisma BU8, No. 11, Lebuh Bandar Utama, 47800, Petaling Jaya, Selangor, Malaysia

    Kim-Yong Ng

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  1. Hariyanti Baharum
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Correspondence to Ikuro Abe or Chai-Ling Ho.

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Baharum, H., Morita, H., Tomitsuka, A. et al. Molecular Cloning, Modeling, and Site-Directed Mutagenesis of Type III Polyketide Synthase from Sargassum binderi (Phaeophyta). Mar Biotechnol 13, 845–856 (2011). https://doi.org/10.1007/s10126-010-9344-5

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