Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
권호 표지
DOI QR코드

DOI QR Code

Functional Characterization of Drosophila melanogaster CYP6A8 Fatty Acid Hydroxylase

  • Sang-A, Lee (Department of Biological Sciences, Konkuk University) ;
  • Vitchan, Kim (Department of Biological Sciences, Konkuk University) ;
  • Byoungyun, Choi (Department of Biological Sciences, Konkuk University) ;
  • Hyein, Lee (College of Pharmacy, Chung Ang University) ;
  • Young-Jin, Chun (College of Pharmacy, Chung Ang University) ;
  • Kyoung Sang, Cho (Department of Biological Sciences, Konkuk University) ;
  • Donghak, Kim (Department of Biological Sciences, Konkuk University)
  • Received : 2022.06.20
  • Accepted : 2022.07.20
  • Published : 2023.01.01
Download PDF
⟨ Previous Next ⟩

Abstract

Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. Here, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni2+-nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type I spectral changes, with Kd values 28 ± 4 and 144 ± 20 µM, respectively. Ultra-performance liquid chromatography-mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a kcat value of 0.038 ± 0.002 min-1 and a Km value of 10 ± 2 µM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a kcat value of 0.135 ± 0.007 min-1 and a Km value of 21 ± 4 µM. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.

Keywords

Acknowledgement

This study was supported by an NRF grant, funded by the Korean government (NRF-2019R1A2C1004722).

References

  1. Adas, F., Salaun, J. P., Berthou, F., Picart, D., Simon, B. and Amet, Y. (1999) Requirement for ω and (ω-1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11. J. Lipid Res. 40, 1990-1997. https://doi.org/10.1016/S0022-2275(20)32422-6
  2. Amichot, M., Tares, S., Brun-Barale, A., Arthaud, L., Bride, J. M. and Berge, J. B. (2004) Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism. Eur. J. Biochem. 271, 1250-1257. https://doi.org/10.1111/j.1432-1033.2004.04025.x
  3. Antoun, J., Amet, Y., Simon, B., Dreano, Y., Corlu, A., Corcos, L., Salaun, J. P. and Plee-Gautier, E. (2006) CYP4A11 is repressed by retinoic acid in human liver cells. FEBS Lett. 580, 3361-3367. https://doi.org/10.1016/j.febslet.2006.05.006
  4. CaJacob, C. A., Chan, W. K., Shephard, E. and Ortiz de Montellano, P. R. (1988) The catalytic site of rat hepatic lauric acid omega-hydroxylase. Protein versus prosthetic heme alkylation in the omega-hydroxylation of acetylenic fatty acids. J. Biol. Chem. 263, 18640-18649. https://doi.org/10.1016/S0021-9258(18)37333-2
  5. Campelo, D., Lautier, T., Urban, P., Esteves, F., Bozonnet, S., Truan, G. and Kranendonk, M. (2017) The hinge segment of human NADPH-cytochrome P450 reductase in conformational switching: the critical role of ionic strength. Front. Pharmacol. 8, 755. https://doi.org/10.3389/fphar.2017.00755
  6. Dunkov, B. C., Guzov, V. M., Mocelin, G., Shotkoski, F., Brun, A., Amichot, M., Ffrench-Constant, R. H. and Feyereisen, R. (1997) The Drosophila cytochrome P450 gene Cyp6a2: structure, localization, heterologous expression, and induction by phenobarbital. DNA Cell Biol. 16, 1345-1356. https://doi.org/10.1089/dna.1997.16.1345
  7. Feyereisen, R. (1999) Insect P450 enzymes. Annu. Rev. Entomol. 44, 507-533. https://doi.org/10.1146/annurev.ento.44.1.507
  8. Feyereisen, R. and Durst, F. (1980) Development of microsomal cytochrome P-450 monooxygenases during the last larval instar of the locust, Locusta migratoria: correlation with the hemolymph 20-hydroxyecdysone titer. Mol. Cell. Endocrinol. 20, 157-169. https://doi.org/10.1016/0303-7207(80)90079-9
  9. Fougeron, A. S., Farine, J. P., Flaven-Pouchon, J., Everaerts, C. and Ferveur, J. F. (2011) Fatty-acid preference changes during development in Drosophila melanogaster. PLoS One 6, e26899. https://doi.org/10.1371/journal.pone.0026899
  10. Guengerich, F. P. (2018) Mechanisms of cytochrome P450-catalyzed oxidations. ACS Catal. 8, 10964-10976. https://doi.org/10.1021/acscatal.8b03401
  11. Hammerer, L., Winkler, C. K. and Kroutil, W. (2018) Regioselective bio-catalytic hydroxylation of fatty acids by cytochrome P450s. Catal. Lett. 148, 787-812. https://doi.org/10.1007/s10562-017-2273-4
  12. He, X., Cryle, M. J., De Voss, J. J. O. and de Montellano, P. R. (2005) Calibration of the channel that determines the omega-hydroxylation regiospecificity of cytochrome P4504A1: catalytic oxidation of 12-HALODOdecanoic acids. J. Biol. Chem. 280, 22697-22705. https://doi.org/10.1074/jbc.M502632200
  13. Helvig, C., Tijet, N., Feyereisen, R., Walker, F. A. and Restifo, L. L. (2004) Drosophila melanogaster CYP6A8, an insect P450 that catalyzes lauric acid (omega-1)-hydroxylation. Biochem. Biophys. Res. Commun. 325, 1495-1502. https://doi.org/10.1016/j.bbrc.2004.10.194
  14. Kim, D., Cha, G.-S., Nagy, L. D., Yun, C.-H. and Guengerich, F. P. (2014) Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11. Biochemistry 53, 6161-6172. https://doi.org/10.1021/bi500710e
  15. Kim, D., Cryle, M. J., De Voss, J. J. and Ortiz de Montellano, P. R. (2007) Functional expression and characterization of cytochrome P450 52A21 from Candida albicans. Arch. Biochem. Biophys. 464, 213-220. https://doi.org/10.1016/j.abb.2007.02.032
  16. Maitra, S., Dombrowski, S. M., Waters, L. C. and Ganguly, R. (1996) Three second chromosome-linked clustered Cyp6 genes show differential constitutive and barbital-induced expression in DDT-resistant and susceptible strains of Drosophila melanogaster. Gene 180, 165-171. https://doi.org/10.1016/S0378-1119(96)00446-5
  17. Miura, Y. (2013) The biological significance of ω-oxidation of fatty acids. Proc. Jpn. Acad. Ser. B 89, 370-382. https://doi.org/10.2183/pjab.89.370
  18. Niwa, R., Matsuda, T., Yoshiyama, T., Namiki, T., Mita, K., Fujimoto, Y. and Kataoka, H. (2004) CYP306A1, a cytochrome P450 enzyme, is essential for ecdysteroid biosynthesis in the prothoracic glands of Bombyx and Drosophila. J. Biol. Chem. 279, 35942-35949. https://doi.org/10.1074/jbc.M404514200
  19. Park, H. G., Lim, Y. R., Eun, C. Y., Han, S., Han, J. S., Cho, K. S., Chun, Y. J. and Kim, D. (2010) Candida albicans NADPH-P450 reductase: expression, purification, and characterization of recombinant protein. Biochem. Biophys. Res. Commun. 396, 534-538. https://doi.org/10.1016/j.bbrc.2010.04.138
  20. Park, H. G., Lim, Y. R., Han, S., Jeong, D. and Kim, D. (2017) Enhanced purification of recombinant rat NADPH-P450 reductase by using a hexahistidine-tag. J. Microbiol. Biotechnol. 27, 983-989. https://doi.org/10.4014/jmb.1701.01028
  21. Plettner, E., Otis, G. W., Wimalaratne, P. D. C., Winston, M. L., Slessor, K. N., Pankiw, T. and Punchihewa, P. W. K. (1997) Species- and caste-determined mandibular gland signals in honeybees (Apis). J. Chem. Ecol. 23, 363-377. https://doi.org/10.1023/B:JOEC.0000006365.20996.a2
  22. Pondeville, E., David, J. P., Guittard, E., Maria, A., Jacques, J. C., Ranson, H., Bourgouin, C. and Dauphin-Villemant, C. (2013) Microarray and RNAi analysis of P450s in Anopheles gambiae male and female steroidogenic tissues: CYP307A1 is required for ecdysteroid synthesis. PLoS One 8, e79861. https://doi.org/10.1371/journal.pone.0079861
  23. Qiu, Y., Tittiger, C., Wicker-Thomas, C., Le Goff, G., Young, S., Wajnberg, E., Fricaux, T., Taquet, N., Blomquist, G. J. and Feyereisen, R. (2012) An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 109, 14858-14863. https://doi.org/10.1073/pnas.1208650109
  24. Seong, K. M., Coates, B. S., Berenbaum, M. R., Clark, J. M. and Pittendrigh, B. R. (2018) Comparative CYP-omic analysis between the DDT-susceptible and -resistant Drosophila melanogaster strains 91-C and 91-R. Pest Manage. Sci. 74, 2530-2543. https://doi.org/10.1002/ps.4936
  25. Tijet, N., Helvig, C. and Feyereisen, R. (2001) The cytochrome P450 gene superfamily in Drosophila melanogaster: annotation, intron-exon organization and phylogeny. Gene 262, 189-198. https://doi.org/10.1016/S0378-1119(00)00533-3
  26. Wang, L., Dankert, H., Perona, P. and Anderson, D. J. (2008) A common genetic target for environmental and heritable influences on aggressiveness in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 105, 5657. https://doi.org/10.1073/pnas.0801327105
  27. Werck-Reichhart, D. and Feyereisen, R. (2000) Cytochromes P450: a success story. Genome Biol. 1, reviews3003.1. https://doi.org/10.1186/gb-2000-1-6-reviews3003