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. 2024 Mar 28;34(3):725-734.
doi: 10.4014/jmb.2310.10018. Epub 2023 Dec 1.

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

Jeong-Hoon Kim  1 , Chan Mi Park  2 , Hae Chan Jeong  1 , Gyeong Han Jeong  3 , Gun Su Cha  4 , Sungbeom Lee  3   5 , Chul-Ho Yun  2   6
Affiliations

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

Jeong-Hoon Kim et al. J Microbiol Biotechnol. .

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-dihydroxy-p-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: CYP102A1 mutant; Cytochrome P450; carbon hydroxylation; cyclic monoterpene; terpinen-4-ol.

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Conflict of interest statement

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Figures

Fig. 1
Fig. 1. Scheme for 1,4-dihydroxy-p-menth-2-ene production by CYP102A1 mutants.
Fig. 2
Fig. 2. Screening of CYP102A1 mutants with terpinen-4-ol.
The mixture contained 0.2 μM of CYP102A1 mutants and 5 mM of terpinen-4-ol in 100 mM potassium phosphate buffer (pH 7.4). The mixture with NGS was incubated for 10 min at 30°C.
Fig. 3
Fig. 3. HPLC chromatogram of terpinen-4-ol and its major products generated by CYP102A1 mutant M179.
The mixture contained 0.2 μM of CYP102A1 M179 and 5 mM of terpinen-4-ol in 100 mM potassium phosphate buffer (pH 7.4). The mixture with NGS was incubated for 1 h at 30°C.
Fig. 4
Fig. 4. LC-MS/MS analysis of terpinen-4-ol and its metabolites produce by CYP102A1 M179.
(A) Analysis of terpinen-4-ol. (C) Analysis of reaction mixture. (B) The MS spectra showed the base peak of terpinen-4-ol is 137.09 m/z. (D) The MS spectra showed the base peak of M1 is 153.07 m/z. (E) The MS spectra showed the base peak of M2 is 153.07 m/z.
Fig. 5
Fig. 5. 1D NMR spectrum of M1.
(A) 1H NMR spectrum of M1 in CDCl3 (600 MHz). (B) 13C NMR spectrum of M1 in CDCl3 (150 MHz).
Fig. 6
Fig. 6. Spectral binding affinity of CYP102A1 M179 toward terpinen-4-ol.
CYP102A1 M179 (2 μM) in 100 mM potassium phosphate buffer was titrated with various substrate concentrations. It was defined as a type II shift. The dissociation constant (Kd) of terpinen-4-ol to CYP102A1 M179 was determined as 0.92 ± 0.26 μM.
Fig. 7
Fig. 7. Time course-dependent formation of terpinen-4-ol metabolites.
(A) The formation of terpinen-4-ol metabolites under 5 mM of terpinen-4-ol. (B) The formation of terpinen-4-ol metabolites under 5 mM of terpinen-4-ol. The reaction mixture of (A) and (B) contained 0.2 μM of CYP102A1 M179 in 100 mM potassium phosphate buffer (pH 7).
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