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.

中文翻译：

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通过细菌 CYP102A1 酶生产 Terpinen-4-ol 单羟基化衍生物。

来自巨大芽孢杆菌的 CYP102A1是生物技术中的重要酶，因为工程化 CYP102A1 酶可以与多种底物反应并产生人类细胞色素 P450 样代谢物。因此，CYP102A1可应用于药物代谢产物的生产。Terpinen-4-ol 是一种环状单萜，是茶树油的主要成分。Terpinen-4-ol 以其治疗作用而闻名，包括抗菌、抗真菌、抗病毒和抗炎。由于萜烯是天然化合物，因此检查新型萜烯并研究萜烯的治疗效果代表了对生态友好化合物的社会需求的响应。在本研究中，我们研究了工程化 CYP102A1 对 terpinen-4-ol 的催化活性。在此测试的 CYP102A1 突变体中，R47L/F81I/F87V/E143G/L188Q/N213S/E267V 突变体对萜品烯-4-醇表现出最高的活性。terpinen-4-ol 的两种主要代谢物是由工程化的 CYP102A1 产生的。主要代谢物的表征通过液相色谱-质谱 (LC-MS)、气相色谱-MS 和核磁共振波谱 (NMR) 进行确认。根据 LC-MS 结果，terpinen-4-ol 与其主要代谢物之间的离子质荷比 (m/z) 相差 16。一种主要代谢物被定义为 1,4-二羟基-通过NMR测定对薄荷-2-烯。鉴于这些结果，我们推测另一种主要代谢物也是单羟基化产物。综上所述，我们建议 CYP102A1 可用于制备新型萜烯衍生物。