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