VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase
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 V...
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MDPI AG
2018-04-01
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| Series: | Molecules |
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| Online Access: | http://www.mdpi.com/1420-3049/23/4/809 |
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| author | Dirk Tischler Ringo Schwabe Lucas Siegel Kristin Joffroy Stefan R. Kaschabek Anika Scholtissek Thomas Heine |
| author_facet | Dirk Tischler Ringo Schwabe Lucas Siegel Kristin Joffroy Stefan R. Kaschabek Anika Scholtissek Thomas Heine |
| author_sort | Dirk Tischler |
| collection | DOAJ |
| description | 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. |
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| language | English |
| last_indexed | 2024-04-12T12:22:30Z |
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| spelling | doaj.art-247a2950a01c400ebbaa58142fbf24952022-12-22T03:33:15ZengMDPI AGMolecules1420-30492018-04-0123480910.3390/molecules23040809molecules23040809VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing MonooxygenaseDirk Tischler0Ringo Schwabe1Lucas Siegel2Kristin Joffroy3Stefan R. Kaschabek4Anika Scholtissek5Thomas Heine6Institute of Biosciences, Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg, GermanyInstitute of Biosciences, Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg, GermanyInstitute of Biosciences, Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg, GermanyInstitute of Biosciences, Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg, GermanyInstitute of Biosciences, Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg, GermanyInstitute of Biosciences, Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg, GermanyInstitute of Biosciences, Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg, GermanyHerein 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.http://www.mdpi.com/1420-3049/23/4/809sulfoxidationepoxidationtwo-component monooxygenaseflavoproteinenantioselective biotransformationfusion proteinprotein linkersoil microorganism |
| spellingShingle | Dirk Tischler Ringo Schwabe Lucas Siegel Kristin Joffroy Stefan R. Kaschabek Anika Scholtissek Thomas Heine VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase Molecules sulfoxidation epoxidation two-component monooxygenase flavoprotein enantioselective biotransformation fusion protein protein linker soil microorganism |
| title | VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase |
| title_full | VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase |
| title_fullStr | VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase |
| title_full_unstemmed | VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase |
| title_short | VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase |
| title_sort | vpstya1 vpstya2b of variovorax paradoxus eps an aryl alkyl sulfoxidase rather than a styrene epoxidizing monooxygenase |
| topic | sulfoxidation epoxidation two-component monooxygenase flavoprotein enantioselective biotransformation fusion protein protein linker soil microorganism |
| url | http://www.mdpi.com/1420-3049/23/4/809 |
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