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Comparison of CYP106A1 and CYP106A2 from Bacillus megaterium – identification of a novel 11-oxidase activity

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Abstract

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.

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Acknowledgments

This work was generously supported by the People Programme (Marie Curie Actions) of the European Union’s 7th Framework Programme (FP7/2007–2013), P4FIFTY–FP7 PEOPLE ITN 2011-289217. The authors thank Wolfgang Reinle and Birgit Heider-Lips for the excellent expression and purification of AdR and Adx4-108 and Nicolas Souza Carmona for the thorough revision of the language.

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The authors declare no conflict of interest.

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Authors and Affiliations

  1. Institute of Biochemistry, Saarland University, Campus B 2.2, 66123, Saarbruecken, Germany

    Flora Marta Kiss, Daniela Schmitz & Rita Bernhardt

  2. Institute of Pharmaceutical Biology, Saarland University, 66123, Saarbruecken, Germany

    Josef Zapp

  3. Institute of Bioanalytical Chemistry, Saarland University, 66123, Saarbruecken, Germany

    Tobias K. F. Dier & Dietrich A. Volmer

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  1. Flora Marta Kiss
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  2. Daniela Schmitz
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  6. Rita Bernhardt
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Correspondence to Rita Bernhardt.

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Kiss, F.M., Schmitz, D., Zapp, J. et al. Comparison of CYP106A1 and CYP106A2 from Bacillus megaterium – identification of a novel 11-oxidase activity. Appl Microbiol Biotechnol 99, 8495–8514 (2015). https://doi.org/10.1007/s00253-015-6563-8

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