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Molecules 2018, 23(4), 809; https://doi.org/10.3390/molecules23040809

VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase

1
Institute of Biosciences, Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg, Germany
2
Microbial Biotechnology, Ruhr University Bochum, Universitätsstr. 150, 44780 Bochum, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Willem van Berkel
Received: 16 March 2018 / Revised: 27 March 2018 / Accepted: 1 April 2018 / Published: 2 April 2018
(This article belongs to the Special Issue Flavoenzymes)
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Abstract

Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein (VpStyA1) and a two domain protein (VpStyA2B) harboring an epoxidase (A2) and a FAD-reductase (B) domain. It was annotated as VpStyA1/VpStyA2B of Variovorax paradoxus EPS. VpStyA2B serves mainly as NADH:FAD-oxidoreductase. A Km of 33.6 ± 4.0 µM for FAD and a kcat of 22.3 ± 1.1 s−1 were determined and resulted in a catalytic efficiency (kcat Km−1) of 0.64 s−1 μM−1. To investigate its NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV) was mutated. One mutant (AAAAA) showed 18.7-fold higher affinity for FAD (kcat Km−1 of 5.21 s−1 μM−1) while keeping wildtype NADH-affinity and -oxidation activity. Both components, VpStyA2B and VpStyA1, showed monooxygenase activity on styrene of 0.14 U mg−1 and 0.46 U mg−1, as well as on benzyl methyl sulfide of 1.62 U mg−1 and 3.11 U mg−1, respectively. The high sulfoxidase activity was the reason to test several thioanisole-like substrates in biotransformations. VpStyA1 showed high substrate conversions (up to 95% in 2 h) and produced dominantly (S)-enantiomeric sulfoxides of all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity. In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase activity. Hence, the linker between the two domains of VpStyA2B has effects on the reductase as well as on the monooxygenase performance. Overall, this monooxygenase represents a promising candidate for biocatalyst development and studying natural fusion proteins. View Full-Text
Keywords: sulfoxidation; epoxidation; two-component monooxygenase; flavoprotein; enantioselective biotransformation; fusion protein; protein linker; soil microorganism sulfoxidation; epoxidation; two-component monooxygenase; flavoprotein; enantioselective biotransformation; fusion protein; protein linker; soil microorganism
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Tischler, D.; Schwabe, R.; Siegel, L.; Joffroy, K.; Kaschabek, S.R.; Scholtissek, A.; Heine, T. VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase. Molecules 2018, 23, 809.

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