Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Production of Mono-Hydroxylated Derivatives of Terpinen-4-ol by Bacterial CYP102A1 Enzymes

  • Jeong-Hoon Kim (School of Biological Sciences and Biotechnology, Graduate School, Chonnam National University) ;
  • Chan Mi Park (School of Biological Sciences and Technology, Chonnam National University) ;
  • Hae Chan Jeong (School of Biological Sciences and Biotechnology, Graduate School, Chonnam National University) ;
  • Gyeong Han Jeong (Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI)) ;
  • Gun Su Cha (Namhae Garlic Research Institute) ;
  • Sungbeom Lee (Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI)) ;
  • Chul-Ho Yun (School of Biological Sciences and Technology, Chonnam National University)
  • Received : 2023.10.13
  • Accepted : 2023.11.22
  • Published : 2024.03.28
Download PDF
⟨ Previous Next ⟩

Abstract

CYP102A1 from Bacillus megaterium is an important enzyme in biotechnology, because engineered CYP102A1 enzymes can react with diverse substrates and produce human cytochrome P450-like metabolites. Therefore, CYP102A1 can be applied to drug metabolite production. Terpinen-4-ol is a cyclic monoterpene and the primary component of essential tea tree oil. Terpinen-4-ol was known for therapeutic effects, including antibacterial, antifungal, antiviral, and anti-inflammatory. Because terpenes are natural compounds, examining novel terpenes and investigating the therapeutic effects of terpenes represent responses to social demands for eco-friendly compounds. In this study, we investigated the catalytic activity of engineered CYP102A1 on terpinen-4-ol. Among CYP102A1 mutants tested here, the R47L/F81I/F87V/E143G/L188Q/N213S/E267V mutant showed the highest activity to terpinen-4-ol. Two major metabolites of terpinen-4-ol were generated by engineered CYP102A1. Characterization of major metabolites was confirmed by liquid chromatography-mass spectrometry (LC-MS), gas chromatography-MS, and nuclear magnetic resonance spectroscopy (NMR). Based on the LC-MS results, the difference in mass-to-charge ratio of an ion (m/z) between terpinen-4-ol and its major metabolites was 16. One major metabolite was defined as 1,4-dihydroxyp-menth-2-ene by NMR. Given these results, we speculate that another major metabolite is also a mono-hydroxylated product. Taken together, we suggest that CYP102A1 can be applied to make novel terpene derivatives.

Keywords

Acknowledgement

This research was funded by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Crop Viruses and Pests Response Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Grant No. 321102-3).

References

  1. Carson CF, Hammer KA, Riley TV. 2006. Melaleuca alternifolia (Tea Tree) oil: a review of antimicrobial and other medicinal properties. Clin. Microbiol. Rev. 19: 50-62.
  2. Homer LE, Leach DN, Lea D, Slade Lee L null, Henry RJ, Baverstock PR. 2000. Natural variation in the essential oil content of Melaleuca alternifolia Cheel (Myrtaceae). Biochem. Syst. Ecol. 28: 367-382.
  3. Schnitzler P, Schon K, Reichling J. 2001. Antiviral activity of Australian tea tree oil and eucalyptus oil against herpes simplex virus in cell culture. Pharmazie 56: 343-347.
  4. Carson CF, Ashton L, Dry L, Smith DW, Riley TV. 2001. Melaleuca alternifolia (tea tree) oil gel (6%) for the treatment of recurrent herpes labialis. J. Antimicrob. Chemother. 48: 450-451.
  5. Carson CF, Riley TV, Cookson BD. 1998. Efficacy and safety of tea tree oil as a topical antimicrobial agent. J. Hosp. Infect. 40: 175-178.
  6. Williams LR, Asre S, Home VN. 1994. Topical applications containing tea tree oil for vaginal conditions. Cosmet. Aerosols Toiletries Aust. 8: 23-26.
  7. Griffin SG, Wyllie SG, Markham JL, Leach DN. 1999. The role of structure and molecular properties of terpenoids in determining their antimicrobial activity. Flavour Fragr. J. 14: 322-332.
  8. Zhang Y, Feng R, Li L, Zhou X, Li Z, Jia R, et al. 2018. The Antibacterial Mechanism of Terpinen-4-ol Against Streptococcus agalactiae. Curr. Microbiol. 75: 1214-1220.
  9. Hammer KA, Carson CF, Riley TV. 1998. In-vitro activity of essential oils, in particular Melaleuca alternifolia (tea tree) oil and tea tree oil products, against Candida spp. J. Antimicrob. Chemother. 42: 591-595.
  10. Hammer KA, Carson CF, Riley TV. 2002. In vitro activity of Melaleuca alternifolia (tea tree) oil against dermatophytes and other filamentous fungi. J. Antimicrob. Chemother. 50: 195-199.
  11. Kong Q, Qi J, An P, Deng R, Meng J, Ren X. 2020. Melaleuca alternifolia oil can delay nutrient damage of grapes caused by aspergillus ochraceus through regulation of key genes and metabolites in metabolic pathways. Postharvest Biol. Technol. 164: 111152.
  12. Burdock GA. 2009. Fenaroli's Handbook of Flavor Ingredients, pp.261. 6th Ed. CRC Press, Boca Raton.
  13. Api AM, Belsito D, Botelho D, Browne D, Bruze M, Burton GA, et al. 2017. RIFM fragrance ingredient safety assessment, 4- Carvomenthenol, CAS Registry Number 562-74-3. Food Chem. Toxicol. 110 Suppl 1: S403-S411.
  14. Bhatia SP, McGinty D, Letizia CS, Api AM. 2008. Fragrance material review on 4-carvomenthenol. Food Chem. Toxicol. 46 Suppl 11: S91-94.
  15. Liao M, Shi S, Wu H, Yang Q, Zhu Z, Xiao J, et al. 2020. Effects of terpinen-4-ol fumigation on protein levels of detoxification enzymes in Tribolium confusum. Arch. Insect Biochem. Physiol. 103: e21653.
  16. Campolo O, Patane V, Verdone AM, Palmeri V. 2012. Survey of solid impurities and active infestation in flours produced in Calabria (Italy). J. Stored Prod. Res. 50: 36-41.
  17. Campolo O, Verdone M, Laudani F, Malacrino A, Chiera E, Palmeri V. 2013. Response of four stored products insects to a structural heat treatment in a flour mill. J. Stored Prod. Res. 54: 54-58.
  18. Cook DJ, Finnigan JD, Cook K, Black GW, Charnock SJ. 2016. Cytochromes P450: history, classes, catalytic mechanism, and industrial application. Adv. Protein Chem. Struct. Biol. 105: 105-126.
  19. Omura T, Sato R. 1962. A new cytochrome in liver microsomes. J. Biol. Chem. 237: 1375-1376.
  20. Omura T, Sato R. 1964. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. J. Biol. Chem. 239: 2370-2378.
  21. Nam W. 2003. 8.12 - Cytochrome P450, pp. 281-307. In McCleverty JA, Meyer TJ (eds.), Comprehensive Coordination Chemistry II. Pergamon, Oxford.
  22. Shankar K, Mehendale HM. 2014. Cytochrome P450, pp. 1125-1127. In Wexler P (ed.), Encyclopedia of Toxicology, 3th Ed. Academic Press, Oxford.
  23. Lynch T, Price A. 2007. The effect of cytochrome P450 metabolism on drug response, interactions, and adverse effects. Am. Fam. 76: 391-396.
  24. Whitehouse CJC, Bell SG, Wong L-L. 2012. P450(BM3) (CYP102A1): connecting the dots. Chem. Soc. Rev. 41: 1218-1260.
  25. Thistlethwaite S, Jeffreys LN, Girvan HM, McLean KJ, Munro AW. 2021. A Promiscuous bacterial P450: the unparalleled diversity of BM3 in pharmaceutical metabolism. Int. J. Mol. Sci. 22: 11380.
  26. Kokorin A, Parshin PD, Bakkes PJ, Pometun AA, Tishkov VI, Urlacher VB. 2021. Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity. Sci. Rep. 11: 21706.
  27. Peters MW, Meinhold P, Glieder A, Arnold FH. 2003. Regio- and enantioselective alkane hydroxylation with engineered cytochromes P450 BM-3. J. Am. Chem. Soc. 125: 13442-13450.
  28. Holec C, Hartrampf U, Neufeld K, Pietruszka J. 2017. P450 BM3-catalyzed regio- and stereoselective hydroxylation aiming at the synthesis of phthalides and isocoumarins. Chembiochem 18: 676-684.
  29. Hui C, Singh W, Quinn D, Li C, Moody TS, Huang M. 2020. Regio- and stereoselectivity in the CYP450BM3-catalyzed hydroxylation of complex terpenoids: a QM/MM study. Phys. Chem. Chem. Phys. 22: 21696-21706.
  30. Huang X, Sun Y, Osawa Y, Chen YE, Zhang H. 2023. Computational redesign of cytochrome P450 CYP102A1 for highly stereoselective omeprazole hydroxylation by UniDesign. J. Biol. Chem. 299: 105050.
  31. Nguyen THH, Woo SM, Nguyen NA, Cha GS, Yeom SJ, Kang HS, et al. 2020. Regioselective hydroxylation of naringin dihydrochalcone to produce neoeriocitrin dihydrochalcone by CYP102A1 (BM3) mutants. Catalysts 10: 823.
  32. Janocha S, Schmitz D, Bernhardt R. 2015. Terpene hydroxylation with microbial cytochrome P450 monooxygenases. Adv. Biochem. Engin./Biotechnol. 148: 215-250.
  33. Urlacher VB, Girhard M. 2012. Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol. 30: 26-36.
  34. Seifert A, Vomund S, Grohmann K, Kriening S, Urlacher VB, Laschat S, et al. 2009. Rational design of a minimal and highly enriched CYP102A1 mutant library with improved regio-, stereo- and chemoselectivity. Chembiochem 10: 853-861.
  35. Seifert A, Antonovici M, Hauer B, Pleiss J. 2011. An efficient route to selective bio-oxidation catalysts: an iterative approach comprising modeling, diversification, and screening, based on CYP102A1. Chembiochem 12: 1346-1351.
  36. Ikebe J, Suzuki M, Komori A, Kobayashi K, Kameda T. 2021. Enzyme modification using mutation site prediction method for enhancing the regioselectivity of substrate reaction sites. Sci. Rep. 11: 19004.
  37. Venkataraman H, Beer SBA de, Geerke DP, Vermeulen NPE, Commandeur JNM. 2012. Regio- and stereoselective hydroxylation of optically active α-ionone enantiomers by engineered cytochrome P450 BM3 mutants. Adv. Synth. Catal. 354: 2172-2184.
  38. Urlacher VB, Makhsumkhanov A, Schmid RD. 2006. Biotransformation of beta-ionone by engineered cytochrome P450 BM-3. Appl. Microbiol. Biotechnol. 70: 53-59.
  39. Maurer SC, Schulze H, Schmid RD, Urlacher V. 2003. Immobilisation of P450 BM-3 and an NADP+ cofactor recycling system: towards a technical application of heme-containing monooxygenases in fine chemical synthesis. Adv. Synth. Catal. 345: 802-810.
  40. Appel D, Lutz-Wahl S, Fischer P, Schwaneberg U, Schmid RD. 2001. A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes. J. Biotechnol. 88: 167-171.
  41. Liu X, Kong JQ. 2017. Steroids hydroxylation catalyzed by the monooxygenase mutant 139-3 from Bacillus megaterium BM3. Acta Pharm. Sin. B. 7: 510-516.
  42. Venkataraman H, Te Poele EM, Rosloniec KZ, Vermeulen N, Commandeur JNM, van der Geize R, et al. 2015. Biosynthesis of a steroid metabolite by an engineered Rhodococcus erythropolis strain expressing a mutant cytochrome P450 BM3 enzyme. Appl. Microbiol. Biotechnol. 99: 4713-4721.
  43. Rea V, Kolkman AJ, Vottero E, Stronks EJ, Ampt K a. M, Honing M, et al. 2012. Active site substitution A82W improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalized by spin relaxation nuclear magnetic resonance studies. Biochemistry 51: 750-760.
  44. Ren Y, Liu S, Jin G, Yang X, Zhou YJ. 2020. Microbial production of limonene and its derivatives: achievements and perspectives. Biotechnol. Adv. 44: 107628.
  45. Hernandez-Ortega A, Vinaixa M, Zebec Z, Takano E, Scrutton NS. 2018. A toolbox for diverse oxyfunctionalisation of monoterpenes. Sci. Rep. 8: 14396.
  46. Sowden RJ, Yasmin S, Rees NH, Bell SG, Wong LL. 2005. Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3. Org. Biomol. Chem. 3: 57-64.
  47. Garcia-Carnelli C, Rodriguez P, Heinzen H, Menendez P. 2014. Influence of culture conditions on the biotransformation of (+)- limonene by Aspergillus niger. Z. Naturforsch. C J. Biosci. 69: 61-67.
  48. Kaspera R, Krings U, Pescheck M, Sell D, Schrader J, Berger RG. 2005. Regio- and stereoselective fungal oxyfunctionalisation of limonenes. Z. Naturforsch. C J. Biosci. 60: 459-466.
  49. Garnes-Portoles F, Lopez-Cruz C, Sanchez-Quesada J, Espinos-Ferri E, Leyva-Perez A. 2022. Solid-catalyzed synthesis of isomers-free terpinen-4-ol. Mol. Catal. 533: 112785.
  50. Retajczyk M, Wroblewska A. 2017. The isomerization of limonene over the Ti-SBA-15 catalyst-The influence of reaction time, temperature, and catalyst content. Catalysts 7: 273.
  51. Haigou R, Miyazawa M. 2012. Metabolism of (+)-terpinen-4-ol by cytochrome P450 enzymes in human liver microsomes. J. Oleo Sci. 61: 35-43.
  52. Miyazawa M, Haigou R. 2011. Determination of cytochrome P450 enzymes involved in the metabolism of (-)-terpinen-4-ol by human liver microsomes. Xenobiotica 41: 1056-1062.
  53. Shapira S, Pleban S, Kazanov D, Tirosh P, Arber N. 2016. Terpinen-4-ol: a novel and promising therapeutic agent for human gastrointestinal cancers. PLoS One 11: e0156540.
  54. Wu CS, Chen YJ, Chen JJW, Shieh JJ, Huang CH, Lin PS, et al. 2012. Terpinen-4-ol induces apoptosis in human nonsmall cell lung cancer in vitro and in vivo. Evid. Based Complement. Altern. Med. 2012: 818261.
  55. Nakayama K, Murata S, Ito H, Iwasaki K, Villareal MO, Zheng YW, et al. 2017. Terpinen-4-ol inhibits colorectal cancer growth via reactive oxygen species. Oncol. Lett. 14: 2015-2024.
  56. Graebin CS, Madeira M de F, Yokoyama-Yasunaka JKU, Miguel DC, Uliana SRB, Benitez D, et al. 2010. Synthesis and in vitro activity of limonene derivatives against Leishmania and Trypanosoma. Eur. J. Med. Chem. 45: 1524-1528.
  57. van Vugt-Lussenburg BMA, Stjernschantz E, Lastdrager J, Oostenbrink C, Vermeulen NPE, Commandeur JNM. 2007. Identification of critical residues in novel drug metabolizing mutants of cytochrome P450 BM3 using random mutagenesis. J. Med. Chem. 50: 455-461.
  58. Carmichael AB, Wong LL. 2001. Protein engineering of Bacillus megaterium CYP102. The oxidation of polycyclic aromatic hydrocarbons. Eur. J. Biochem. 268: 3117-3125.
  59. Cao NT, Cha GS, Kim JH, Lee Y, Yun CH, Nguyen NA. 2023. Production of an O-desmethylated product, a major human metabolite, of rabeprazole sulfide by bacterial P450 enzymes. Enzyme Microb. Technol. 171: 110328.
  60. Jang HH, Ryu SH, Le TK, Doan TTM, Nguyen THH, Park KD, et al. 2017. Regioselective C-H hydroxylation of omeprazole sulfide by Bacillus megaterium CYP102A1 to produce a human metabolite. Biotechnol. Lett. 39: 105-112.
  61. Rudback J, Bergstrom MA, Borje A, Nilsson U, Karlberg AT. 2012. α-Terpinene, an antioxidant in tea tree oil, autoxidizes rapidly to skin allergens on air exposure. Chem. Res. Toxicol. 25: 713-721.
  62. Valente P, Avery TD, Taylor DK, Tiekink ERT. 2009. Synthesis and chemistry of 2,3-dioxabicyclo[2.2.2]octane-5,6-diols. J. Org. Chem. 74: 274-282.
  63. Ahmed AA. 2000. Highly oxygenated monoterpenes from Chenopodium ambrosioides. J. Nat. Prod. 63: 989-991.
  64. Li J, Gu W, Wang Z, Zhou X, Chen Y. 2023. Asymmetric bio-epoxidation of unactivated alkenes. Chembiochem 24: e202200719.