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Mutation study of conserved amino acid residues of Spirulina Δ6-acyl-lipid desaturase showing involvement of histidine 313 in the regioselectivity of the enzyme

  • Applied Genetics and Molecular Biotechnology
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Abstract

In the cyanobacterium Spirulina platensis, the desaturation process is carried out by three desaturases: the Δ9, Δ12 and Δ6 desaturases, encoded by desC, desA and desD, respectively. The Δ6 desaturase is responsible for the catalysis of linoleic acid, yielding γ-linolenic acid (18:3Δ9,12,6), the end-product of the process. In this study, the desD gene was expressed in Escherichia coli using a pTrcHisA expression system. In order to identify the amino acid residues involved in the enzymatic activity, a sequence comparison was performed using various organisms. The alignment revealed three conserved histidine clusters, a number of conserved residues among all listed organisms and a few conserved residues among cyanobacterial species possibly involved in the desaturation activity. A series of site-directed mutations were generated in the desD gene to evaluate the role of these residues vis-à-vis the enzyme function. This approach revealed that: (1) H313 is involved in the regioselectivity of the enzyme, (2) the three histidine clusters together with H313, H315, D138 and E140 are required for enzymatic activity, most likely as providers of the catalytic Fe center and (3) W294 is also essential for the activity of Δ6 desaturase, possibly by forming part of the substrate-binding pocket.

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References

  • Avelange-Macherel M, Macherel D, Wada H, Murata N (1991) Site-directed mutagenesis of histidine residues in the delta 12 acyl-lipid desaturase of Synechocystis. FEBS 361:111–114

    Article  Google Scholar 

  • Bloomfield DK, Bloch K (1960) Formation of unsaturated fatty acids. J Biol Chem 235:337–345

    CAS  PubMed  Google Scholar 

  • Broadwater JA, Whittle E, Shanklin J (2002) Desaturation and hydroxylation; residue 148 and 324 of Arabidopsis FAD2, in addition to substrate chain length, exert a major influence in partitioning of catalytic specificity. Biol Chem 277:15613–15620

    Article  CAS  Google Scholar 

  • Cahoon EB, Mills LA, Shanklin J (1996) Modification of the fatty acid composition of Escherichia coli by coexpression of a plant acyl–acyl carrier protein desaturase and ferredoxin. J Bacteriol 178:936–939

    CAS  PubMed  Google Scholar 

  • Cahoon EB, Coughlan SJ, Shanklin J (1997) Characterization of a structurally and functionally diverged acyl–acyl carrier protein desaturase from milkweed seed. Plant Mol Biol 33:1105–1110

    Article  CAS  PubMed  Google Scholar 

  • Cahoon EB, Lindqvist Y, Schneider G, Shanklin J (1997) Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position. Proc Natl Acad Sci USA 94:4872–4877

    Article  CAS  PubMed  Google Scholar 

  • Cahoon EB, Shah S, Shanklin J, Browse J (1998) A determinant of substrate specificity predicted from the acyl–acyl carrier protein desaturase of developing cat’s claw seed. Plant Physiol 117:593–598

    Article  CAS  PubMed  Google Scholar 

  • Creighton TE (1993) Proteins: structures and molecular properties. Freeman, New York

  • Deng WP, Nickoloff JA (1992) Site directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem 200:81–88

    CAS  PubMed  Google Scholar 

  • Deshnium P, Paithoonrangsarid K, Suphratrakul A, Meesapyodsuk D, Tanticharoen M, Cheevadhanarak S (2000) Temperature-independent and dependent expression of desaturase genes in filamentous cyanobacterium Spirulina platensis C1 (Arthrospira sp. PCC 9438). FEMS Microbiol Lett 184:207–213

    Article  CAS  PubMed  Google Scholar 

  • Diaz AR, Mansilla MC, Vila AJ, Mendoza D (2002) Membrane topology of the acyl-lipid desaturase from Bacillus subtilis. Biol Chem 277:48099–48106

    Article  CAS  Google Scholar 

  • Esfahani M, Barnes EMJ, Wakil S (1969) Control of fatty acid composition in phospholipids of Escherichia coli: response to fatty acid supplements in a fatty acid auxotroph. Proc Natl Acad Sci USA 64:1057–1064

    CAS  PubMed  Google Scholar 

  • Fay L, Richli U (1991) Location of double bonds in polyunsaturated fatty acids by gas chromatography-mass spectrometry after 4,4-dimethyloxazoline derivatization. J Chromatogr 541:89–98

    Article  CAS  Google Scholar 

  • Gravel P, Golaz O (1996) Protein blotting by the semi-dry method. In: Walker JM (ed) The protein protocols handbook. Humana, New Jersey, pp 249–260

  • Holloway PW (1983) Fatty acid desaturation. In Boyer PD (ed) The enzymes, vol 16. Academic, Orlando, pp 63–83

  • Hongsthong A, Deshnium P, Paithoonrangsarid K, Cheevadhanarak S, Tanticharoen M (2003) Differential responses of the three acyl-lipid desaturases to immediate temperature reduction occurred in two lipid membranes of Spirulina platensis strain C1. J Biosci Bioeng 96:519–524

    Article  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:269–275

    Google Scholar 

  • Mcguire KA, Siggaard-Andersen M, Bangera MG, Olsen JG, Wettstein-Knowles P (2001) β-Ketoacyl-(acyl carrier protein) synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket. Biochemistry 40:9836–9845

    Article  CAS  PubMed  Google Scholar 

  • Murata N, Nishida I (1987) Lipids of blue-green algae. In: Stumpf PK (ed) The biochemistry of plants, vol 9. Academic, Orlando, pp 315–347

  • Murata N, Wada H (1995) Acyl-lipid desaturases and their importance in the tolerance and acclimation to cold of cyanobacteria. Biochem J 308:1–8

    CAS  PubMed  Google Scholar 

  • Murata N, Deshnium P, Tasaka Y (1996) Biosynthesis of γ-linolenic acid in the cyanobacterium Spirulina platensis. In: Huang YS, Mills DE (eds) γ-Linolenic acid metabolism and its roles in nutrition and medicine. AOCS, Urbana-Champaign, pp 22–32

  • Mustady L, Los DA, Gombos Z, Murata N (1996) Immunocytochemical location of acyl-lipid desaturases in cyanobacterial cells: evidence that both thylakoid membranes and cytoplasmic membranes are the sites for lipid desaturation. Proc Natl Acad Sci USA 93:10524–10527

    Article  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Schmidt H, Heinz E (1993) Direct desaturation of intact galactolipids by a desaturase solubilized from spinach (Spinacia oleracea) chloroplast envelopes. Biochem J 289:777–782

    CAS  PubMed  Google Scholar 

  • Schmidt H, Sperling P, Heinz E (1993) Biochemistry and molecular biology of membrane and storage lipids of plants. American Society for Plant Physiologists, Rockville

  • Schultz DJ, Chung Suh M, Ohlrogge JB (2000) Stearoyl–acyl carrier protein and unusual acyl–acyl carrier protein desaturase activities are differentially influences by ferredoxin. Plant Physiol 124:681–692

    Article  CAS  PubMed  Google Scholar 

  • Shanklin J, Cahoon EB (1998) Desaturation and related modifications of fatty acids. Annu Rev Plant Physiol Plant Mol Biol 49:611–641

    Article  CAS  PubMed  Google Scholar 

  • Shanklin J, Whittle E, Fox BG (1994) Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, steroyl-CoA desaturase and are conserved in alkene hydroxylase and xylene monooxygenase. Biochemistry 33:12787–12794

    CAS  PubMed  Google Scholar 

  • Sperling P, Ternes P, Zank TK, Heinz E (2003) The evolution of desaturases. Prostaglandins Leukot Essent Fatty Acids 68:73–95

    Article  CAS  PubMed  Google Scholar 

  • Sundberg RJ, Martin RB (1974) Interactions of histidine and other imidazole derivatives with transition metal ions in chemical and biological systems. Chem Rev 74:471–517

    CAS  Google Scholar 

  • Ta TD, Vickery LE (1992) Cloning, sequencing and overexpression of a [2Fe–2S] ferredoxin gene from Escherichia coli. J Biol Chem 267:11120–11125

    CAS  PubMed  Google Scholar 

  • Tocher DR, Leaver MJ, Hodgson PA (1998) Recent advances in the biochemistry and molecular biology of fatty acyl desaturases. Prog Lipid Res 37:73–117

    Article  CAS  PubMed  Google Scholar 

  • Wada H, Avelange-Macherel M, Murata N (1993) The desA gene of the cyanobacterium Synechocystis sp. strain PCC 6803 is the structural gene for Δ12 desaturase. J Bacteriol 175:6056–6058

    CAS  PubMed  Google Scholar 

  • Whittle E, Shanklin J (2001) Engineering delta9-16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor delta9-18:0-ACP desaturase. Biol Chem 276:21500–21505

    Article  CAS  Google Scholar 

  • Zámocky M, Gunther R, Jakopitsch C, Obinger C (2001) The molecular peculiarities of catalase–peroxidase. FEBS 492:177–182

    Article  Google Scholar 

Download references

Acknowledgements

This research was funded by a grant from the National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok, Thailand. The experiments comply with the current laws of Thailand.

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Authors and Affiliations

  1. Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetic Engineering and Biotechnology, King Mongkut’s University of Technology–Thonburi (Bangkhuntien), 83 Moo8, Thakham, Bangkhuntien, Bangkok, 10150, Thailand

    Apiradee Hongsthong & Sanjukta Subudhi

  2. School of Bioresources and Technology, King Mongkut’s University of Technology–Thonburi (Bangkhuntien), 83 Moo8, Thakham, Bangkhuntien, Bangkok, 10150, Thailand

    Matura Sirijuntarat & Supapon Cheevadhanarak

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  1. Apiradee Hongsthong
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  2. Sanjukta Subudhi
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  3. Matura Sirijuntarat
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  4. Supapon Cheevadhanarak
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Correspondence to Apiradee Hongsthong.

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Hongsthong, A., Subudhi, S., Sirijuntarat, M. et al. Mutation study of conserved amino acid residues of Spirulina Δ6-acyl-lipid desaturase showing involvement of histidine 313 in the regioselectivity of the enzyme. Appl Microbiol Biotechnol 66, 74–84 (2004). https://doi.org/10.1007/s00253-004-1655-x

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  • DOI: https://doi.org/10.1007/s00253-004-1655-x

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