Abstract
Polyunsaturated fatty acids (PUFAs) are essential lipids for cell function, normal growth, and development, serving as key structural components of biological membranes and modulating critical signal transduction events. Omega-3 (n-3) long chain PUFAs (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect against inflammatory diseases and enhance brain development and function. This had led to a marked increase in demand for fish and fish oils in human diets, supplements, and aquaculture and created a need for new, sustainable n-3 LC-PUFA sources. We have studied for the first time homogenous preparations of the membrane-type ω6 and ω3 fatty acid desaturases from the fungus Mortierella alpina, as a model system to produce PUFAs. These desaturases possess a di-iron metal center and are selective for 18:1 n-9 and 18:2 n-6 acyl-CoA substrates, respectively. Sequence alignments and membrane topology predictions support that these enzymes have unique cap regions that may include the rearrangement and repositioning of the active site, especially when compared to the mammalian stearoyl–coenzyme A desaturase-1 (SCD1) and the related sphingolipid α-hydroxylase (Scs7p) that act upon different substrates.
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References
Bai Y, McCoy JG, Levin EJ, Sobrado P, Rajashankar KR, Fox BG, Zhou M (2015) X-ray structure of a mammalian stearoyl-CoA desaturase. Nature 524(7564):252–256. https://doi.org/10.1038/nature14549
Bazinet RP, Laye S (2014) Polyunsaturated fatty acids and their metabolites in brain function and disease. Nat Rev Neurosci 15(12):771–785. https://doi.org/10.1038/nrn3820
Chen C, Huang Q-L, Jiang S-H, Pan X, Hua Z-C (2006) Immobilized protein ZZ, an affinity tool for immunoglobulin isolation and immunological experimentation. Biotechnol Appl Biochem 45(2):87–92. https://doi.org/10.1042/BA20060055
Chen H, Gu Z, Zhang H, Wang M, Chen W, Lowther WT, Chen YQ (2013) Expression and purification of integral membrane fatty acid desaturases. PLoS One 8(3):e58139. https://doi.org/10.1371/journal.pone.0058139
Enoch HG, Catalá A, Strittmatter P (1976) Mechanism of rat liver microsomal stearyl-CoA desaturase. Studies of the substrate specificity, enzyme-substrate interactions, and the function of lipid. J Biol Chem 251(16):5095–5103
Fox BG, Shanklin J, Somerville C, Munck E (1993) Stearoyl-acyl carrier protein delta 9 desaturase from Ricinus communis is a diiron-oxo protein. Proc Natl Acad Sci U S A 90(6):2486–2490. https://doi.org/10.1073/pnas.90.6.2486
Goren MA, Fox BG (2008) Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex. Protein Expr Purif 62(2):171–178. https://doi.org/10.1016/j.pep.2008.08.002
Gu Z, Shan K, Chen H, Chen YQ (2015) n-3 polyunsaturated fatty acids and their role in cancer chemoprevention. Curr Pharmacol Rep 1(5):283–294. https://doi.org/10.1007/s40495-015-0043-9
Guy JE, Whittle E, Kumaran D, Lindqvist Y, Shanklin J (2007) The crystal structure of the ivy Δ4-16:0-ACP desaturase reveals structural details of the oxidized active site and potential determinants of regioselectivity. J Biol Chem 282(27):19863–19871. https://doi.org/10.1074/jbc.M702520200
Käll L, Krogh A, Sonnhammer EL (2005) An HMM posterior decoder for sequence feature prediction that includes homology information. Bioinformatics 21(Suppl 1):i251–i257. https://doi.org/10.1093/bioinformatics/bti1014
Kainou K, Kamisaka Y, Kimura K, Uemura H (2006) Isolation of delta12 and omega3-fatty acid desaturase genes from the yeast Kluyveromyces lactis and their heterologous expression to produce linoleic and alpha-linolenic acids in Saccharomyces cerevisiae. Yeast 23(8):605–612. https://doi.org/10.1002/yea.1378
Kurian JR, Bajad SU, Miller JL, Chin NA, Trepanier LA (2004) NADH cytochrome b5 reductase and cytochrome b5 catalyze the microsomal reduction of xenobiotic hydroxylamines and amidoximes in humans. J Pharmacol Exp Ther 311(3):1171–1178. https://doi.org/10.1124/jpet.104.072389
Liu KE, Valentine AM, Wang DL, Huynh BH, Edmondson DE, Salifoglou A, Lippard SJ (1995) Kinetic and spectroscopic characterization of intermediates and component interactions in reactions of methane monooxygenase from Methylococcus capsulatus (Bath). J Am Chem Soc 117(41):10174–10185. https://doi.org/10.1021/ja00146a002
Moche M, Shanklin J, Ghoshal A, Lindqvist Y (2003) Azide and acetate complexes plus two iron-depleted crystal structures of the di-iron enzyme delta9 stearoyl-acyl carrier protein desaturase. Implications for oxygen activation and catalytic intermediates. J Biol Chem 278(27):25072–25080. https://doi.org/10.1074/jbc.M301662200
Oshino N, Imai Y, Sato R (1971) A function of cytochrome b5 in fatty acid desaturation by rat liver microsomes. J Biochem 69(1):155–167
Pikus JD, Studts JM, Achim C, Kauffmann KE, Munck E, Steffan RJ, McClay K, Fox BG (1996) Recombinant toluene-4-monooxygenase: catalytic and Mössbauer studies of the purified diiron and rieske components of a four-protein complex. Biochemistry 35(28):9106–9119. https://doi.org/10.1021/bi960456m
Reeb J, Kloppmann E, Bernhofer M, Rost B (2015) Evaluation of transmembrane helix predictions in 2014. Proteins 83(3):473–484. https://doi.org/10.1002/prot.24749
Sakuradani E, Abe T, Iguchi K, Shimizu S (2005) A novel fungal ω3-desaturase with wide substrate specificity from arachidonic acid-producing Mortierella alpina 1S-4. Appl Microbiol Biotechnol 66(6):648–654. https://doi.org/10.1007/s00253-004-1760-x
Sakuradani E, Kobayashi M, Ashikari T, Shimizu S (1999) Identification of Delta12-fatty acid desaturase from arachidonic acid-producing mortierella fungus by heterologous expression in the yeast Saccharomyces cerevisiae and the fungus Aspergillus oryzae. Eur J Biochem 261(3):812–820
Santomartino R, Riego-Ruiz L, Bianchi MM (2017) Three, two, one yeast fatty acid desaturases: regulation and function. World J Microbiol Biotechnol 33(5):89. https://doi.org/10.1007/s11274-017-2257-y
Shanklin J, Guy JE, Mishra G, Lindqvist Y (2009) Desaturases: emerging models for understanding functional diversification of diiron-containing enzymes. J Biol Chem 284(28):18559–18563. https://doi.org/10.1074/jbc.R900009200
Shanklin J, Whittle E (2003) Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family. FEBS Lett 545(2–3):188–192
Shanklin J, Whittle E, Fox BG (1994) Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase. Biochemistry 33(43):12787–12794. https://doi.org/10.1021/bi00209a009
Shi H, Chen H, Gu Z, Song Y, Zhang H, Chen W, Chen YQ (2015) Molecular mechanism of substrate specificity for delta 6 desaturase from Mortierella alpina and Micromonas pusilla. J Lipid Res 56(12):2309–2321. https://doi.org/10.1194/jlr.M062158
Shimiziu S, Kawashima H, Shinmen Y, Akimoto K, Yamada H (1988) Production of eicosapentaenoic acid by Mortierella fungi. J Am Oil Chem Soc 65(9):1455–1459. https://doi.org/10.1007/BF02898307
Shinmen Y, Shimizu S, Akimoto K, Kawashima H, Yamada H (1989) Production of arachidonic acid by Mortierella fungi. Appl Microbiol Biotechnol 31(1):11–16. https://doi.org/10.1007/BF00252518
Strittmatter P, Rogers MJ, Spatz L (1972) The binding of cytochrome b5 to liver microsomes. J Biol Chem 247(22):7188–7194
Strittmatter P, Spatz L, Corcoran D, Rogers MJ, Setlow B, Redline R (1974) Purification and properties of rat liver microsomal stearyl coenzyme A desaturase. Proc Natl Acad Sci U S A 71(11):4565–4569. https://doi.org/10.1073/pnas.71.11.4565
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729. https://doi.org/10.1093/molbev/mst197
Wang H, Klein MG, Zou H, Lane W, Snell G, Levin I, Li K, Sang BC (2015) Crystal structure of human stearoyl-coenzyme A desaturase in complex with substrate. Nat Struct Mol Biol 22(7):581–585. https://doi.org/10.1038/nsmb.3049
Wang L, Chen W, Feng Y, Ren Y, Gu Z, Chen H, Wang H, Thomas MJ, Zhang B, Berquin IM, Li Y, Wu J, Zhang H, Song Y, Liu X, Norris JS, Wang S, Du P, Shen J, Wang N, Yang Y, Wang W, Feng L, Ratledge C, Zhang H, Chen YQ (2011) Genome characterization of the oleaginous fungus Mortierella alpina. PLoS One 6(12):e28319. https://doi.org/10.1371/journal.pone.0028319
Wang M, Chen H, Gu Z, Zhang H, Chen W, Chen YQ (2013) ω3 fatty acid desaturases from microorganisms: structure, function, evolution, and biotechnological use. Appl Microbiol Biotechnol 97(24):10255–10262. https://doi.org/10.1007/s00253-013-5336-5
Weylandt KH, Chen YQ, Lim K, HM S, Cittadini A, Calviello G (2015) ω-3 PUFAs in the prevention and cure of inflammatory, degenerative, and neoplastic diseases 2014. Biomed Res Int 2015:695875. https://doi.org/10.1155/2015/695875
Whittle E, Cahoon EB, Subrahmanyam S, Shanklin J (2005) A multifunctional acyl-acyl carrier protein desaturase from Hedera helix L. (English ivy) can synthesize 16- and 18-carbon monoene and diene products. J Biol Chem 280(31):28169–28176. https://doi.org/10.1074/jbc.M504205200
Zhu G, Koszelak-Rosenblum M, Connelly SM, Dumont ME, Malkowski MG (2015) The crystal structure of an integral membrane fatty acid α-hydroxylase. J Biol Chem 290(50):29820–29833. https://doi.org/10.1074/jbc.M115.680124
Acknowledgements
We thank Lihong Shi, Jill Clodfelter, and Aaron Graff for their technical support. We thank Dr. Edward Pryor for his advice in the design of the expression constructs.
Funding
This research was supported by the National Natural Science Foundation of China (NSFC) (31722041, 21276108), the China Scholarship Council, the Fundamental Research Funds for the Central Universities (JUSRP51702A), the Crystallography and Computational Biophysics Shared Resource of the Comprehensive Cancer Center of Wake Forest Baptist Medical Center (NCI CCSG P30CA012197) and Collaborative innovation center of food safety and quality control in Jiangsu Province.
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Wang, M., Chen, H., Ailati, A. et al. Substrate specificity and membrane topologies of the iron-containing ω3 and ω6 desaturases from Mortierella alpina . Appl Microbiol Biotechnol 102, 211–223 (2018). https://doi.org/10.1007/s00253-017-8585-x
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DOI: https://doi.org/10.1007/s00253-017-8585-x