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Characterization of a recombinant 7,8-linoleate diol synthase from Glomerella cingulate

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Abstract

A putative diol synthase from the fungus Glomerella cingulate was cloned and expressed in Escherichia coli. The putative diol synthase from G. cingulate was purified by His-Trap affinity chromatography with a specific activity of 0.87 U mg−1, an eightfold purification, and a yield of 28 %. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 7,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid (7,8-DiHODE) per min. The purified enzyme was estimated as a 127-kDa tetramer with a molecular mass of 510 kDa by gel filtration chromatography. The enzyme converted linoleic acid to a product, identified as 7S,8S-DiHODE by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (k cat/K m) of 7,8-diol synthase from G. cingulate for the conversion of fatty acid to dihydroxy fatty acid followed the order linoleic acid > α-linolenic acid > oleic acid > palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). The activity of the enzyme for the conversion of 7,8-DiHODE from linoleic acid was maximal at pH 6.5, 40 °C, and 2.5 % (v/v) dimethyl sulfoxide (DMSO). Under these conditions, 7,8-LDS from G. cingulate converted 1.0 mM linoleic acid to 0.62 mM 7,8-DiHODE for 30 min, with a conversion yield of 62 % (mol/mol), via 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE) as an intermediate. The accumulation of 8-HPODE was due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain.

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References

  • Andreou A, Brodhun F, Feussner I (2009) Biosynthesis of oxylipins in non-mammals. Prog Lipid Res 48:148–170

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brodhun F, Gobel C, Hornung E, Feussner I (2009) Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450. J Biol Chem 284:11792–11805

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brodowsky ID, Hamberg M, Oliw EH (1992) A linoleic acid (8R)-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis. Biosynthesis of (8R)-hydroxylinoleic acid and (7S,8S)-dihydroxylinoleic acid from (8R)-hydroperoxylinoleic acid. J Biol Chem 267:14738–14745

    CAS  PubMed  Google Scholar 

  • Cary JW, Harris-Coward PY, Ehrlich KC, Moore GG, Wei Q, Bhatnagar D (2012) Functional and phylogenetic analysis of the Aspergillus ochraceoroseus aflQ (ordA) gene ortholog. Mycologia 104:857–864

    Article  CAS  PubMed  Google Scholar 

  • Estupinan M, Diaz P, Manresa A (2014) Unveiling the genes responsible for the unique Pseudomonas aeruginosa oleate-diol synthase activity. Biochim Biophys Acta 1842:1360–1371

    Article  PubMed  Google Scholar 

  • Garscha U, Jernerén F, Chung D, Keller NP, Hamberg M, Oliw EH (2007) Identification of dioxygenases required for Aspergillus development. Studies of products, stereochemistry, and the reaction mechanism. J Biol Chem 282:34707–34718

    Article  CAS  PubMed  Google Scholar 

  • Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345

    Article  CAS  PubMed  Google Scholar 

  • Hoffmann I, Oliw EH (2013) 7,8- and 5,8-Linoleate diol synthases support the heterolytic scission of oxygen-oxygen bonds by different amide residues. Arch Biochem Biophys 539:87–91

    Article  CAS  PubMed  Google Scholar 

  • Hoffmann I, Jernerén F, Garscha U, Oliw EH (2011) Expression of 5,8-LDS of Aspergillus fumigatus and its dioxygenase domain. A comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase. Arch Biochem Biophys 506:216–222

    Article  CAS  PubMed  Google Scholar 

  • Hoffmann I, Jernerén F, Oliw EH (2013) Expression of fusion proteins of Aspergillus terreus reveals a novel allene oxide synthase. J Biol Chem 288:11459–11469

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jeong YJ, Seo MJ, Shin KC, Oh DK (2015) Production of 8-hydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by recombinant cells expressing H1004A–C1006S variant of Aspergillus nidulans diol synthase. J Mol Catal B Enzym 115:35–42

    Article  CAS  Google Scholar 

  • Jernerén F, Garscha U, Hoffmann I, Hamberg M, Oliw EH (2010a) Reaction mechanism of 5,8-linoleate diol synthase, 10R-dioxygenase, and 8,11-hydroperoxide isomerase of Aspergillus clavatus. Biochim Biophys Acta 1801:503–507

    Article  PubMed  Google Scholar 

  • Jernerén F, Sesma A, Franceschetti M, Hamberg M, Oliw EH (2010b) Gene deletion of 7,8-linoleate diol synthase of the rice blast fungus: studies on pathogenicity, stereochemistry, and oxygenation mechanisms. J Biol Chem 285:5308–5316

    Article  PubMed  PubMed Central  Google Scholar 

  • Oliw EH, Su C, Skogstrom T, Benthin G (1998) Analysis of novel hydroperoxides and other metabolites of oleic, linoleic, and linolenic acids by liquid chromatography-mass spectrometry with ion trap MSn. Lipids 33:843–852

    Article  CAS  PubMed  Google Scholar 

  • Sangeetha C, Rawal R (2008) Nutritional studies of Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. the incitant of mango anthracnose. World J Agric Sci 4:717–720

    Google Scholar 

  • Seo MJ, Shin KC, Oh DK (2014) Production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by whole recombinant Escherichia coli cells expressing diol synthase from Aspergillus nidulans. Appl Microbiol Biotechnol 98:7447–7456

    Article  CAS  PubMed  Google Scholar 

  • Su C, Oliw EH (1996) Purification and characterization of linoleate 8-dioxygenase from the fungus Gaeumannomyces graminis as a novel hemoprotein. J Biol Chem 271:14112–14118

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsitsigiannis DI, Kowieski TM, Zarnowski R, Keller NP (2004a) Endogenous lipogenic regulators of spore balance in Aspergillus nidulans. Eukaryotic Cell 3:1398–1411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsitsigiannis DI, Zarnowski R, Keller NP (2004b) The lipid body protein, PpoA, coordinates sexual and asexual sporulation in Aspergillus nidulans. J Biol Chem 279:11344–11353

    Article  CAS  PubMed  Google Scholar 

  • Wadman MW, de Vries RP, Kalkhove SI, Veldink GA, Vliegenthart JF (2009) Characterization of oxylipins and dioxygenase genes in the asexual fungus Aspergillus niger. BMC Microbiol 9:59

    Article  PubMed  PubMed Central  Google Scholar 

  • Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112:531–552

    CAS  PubMed  Google Scholar 

  • Zhao B, Lei L, Kagawa N, Sundaramoorthy M, Banerjee S, Nagy LD, Guengerich FP, Waterman MR (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J Biol Chem 287:10613–10622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This paper was supported by Konkuk University in 2015.

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Authors and Affiliations

  1. Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea

    Min-Ju Seo, Kyung-Chul Shin, Jung-Ung An, Woo-Ri Kang & Deok-Kun Oh

  2. National Center for Inter-University Research Facilities (NCIRF), Seoul National University, Seoul, 08826, Republic of Korea

    Yoon-Joo Ko

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  1. Min-Ju Seo
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  2. Kyung-Chul Shin
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  4. Woo-Ri Kang
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Correspondence to Deok-Kun Oh.

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This study was funded by Konkuk University.

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Seo, MJ., Shin, KC., An, JU. et al. Characterization of a recombinant 7,8-linoleate diol synthase from Glomerella cingulate . Appl Microbiol Biotechnol 100, 3087–3099 (2016). https://doi.org/10.1007/s00253-015-7132-x

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  • DOI: https://doi.org/10.1007/s00253-015-7132-x

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