Kiss Flora Marta, Schmitz Daniela, Zapp Josef, Dier Tobias K F, Volmer Dietrich A, Bernhardt Rita
Institute of Biochemistry, Saarland University, Campus B 2.2, 66123, Saarbruecken, Germany.
Institute of Pharmaceutical Biology, Saarland University, 66123, Saarbruecken, Germany.
Appl Microbiol Biotechnol. 2015 Oct;99(20):8495-514. doi: 10.1007/s00253-015-6563-8. Epub 2015 Apr 24.
The CYP106A subfamily hydroxylates steroids, diterpenes, and triterpenes in a regioselective and stereoselective manner, which is a challenging task for synthetic chemistry. The well-studied CYP106A2 enzyme, from the Bacillus megaterium strain ATCC 13368, is a highly promising candidate for the pharmaceutical industry. It shares 63 % amino acid sequence identity with CYP106A1 from B. megaterium DSM319, which was recently characterized. A focused steroid library was screened with both CYP106A1 and CYP106A2. Out of the 23 tested steroids, 19 were successfully converted by both enzymes during in vitro and in vivo reactions. Thirteen new substrates were identified for CYP106A1, while the substrate spectrum of CYP106A2 was extended by seven new members. Finally, six chosen steroids were further studied on a preparative scale employing a recombinant B. megaterium MS941 whole-cell system, yielding sufficient amounts of product for structure characterization by nuclear magnetic resonance. The hydroxylase activity was confirmed at positons 6β, 7β, 9α, and 15β. In addition, the CYP106A subfamily showed unprecedented 11-oxidase activity, converting 11β-hydroxysteroids to their 11-keto derivatives. This novel reaction and the diverse hydroxylation positions on pharmaceutically relevant compounds underline the role of the CYP106A subfamily in drug development and production.
CYP106A亚家族以区域选择性和立体选择性方式使甾体、二萜和三萜发生羟基化反应，这对合成化学来说是一项具有挑战性的任务。深入研究的来自巨大芽孢杆菌菌株ATCC 13368的CYP106A2酶是制药行业极具潜力的候选对象。它与最近已被表征的巨大芽孢杆菌DSM319的CYP106A1具有63%的氨基酸序列同一性。用CYP106A1和CYP106A2对一个聚焦甾体文库进行了筛选。在23种测试的甾体中，有19种在体外和体内反应中均被这两种酶成功转化。为CYP106A1鉴定出了13种新底物，而CYP106A2的底物谱增加了7个新成员。最后，使用重组巨大芽孢杆菌MS941全细胞系统对六种选定的甾体进行了制备规模的进一步研究，得到了足够量的产物用于通过核磁共振进行结构表征。在6β、7β、9α和15β位确认了羟化酶活性。此外，CYP106A亚家族表现出前所未有的11-氧化酶活性，可将11β-羟基甾体转化为其11-酮衍生物。这种新反应以及在药学相关化合物上多样的羟基化位置突显了CYP106A亚家族在药物开发和生产中的作用。
