VpStyA1/VpStyA2B of variovorax paradoxus EPS: An aryl alkyl sulfoxidase rather than a styrene epoxidizing monooxygenase
Publikation: Beitrag in Fachzeitschrift › Forschungsartikel › Beigetragen › Begutachtung
Beitragende
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.
Details
Originalsprache | Englisch |
---|---|
Aufsatznummer | 809 |
Fachzeitschrift | Molecules |
Jahrgang | 23 |
Ausgabenummer | 4 |
Publikationsstatus | Veröffentlicht - 2018 |
Peer-Review-Status | Ja |
Extern publiziert | Ja |
Externe IDs
PubMed | 29614810 |
---|---|
ORCID | /0000-0002-7109-2788/work/172571672 |
Schlagworte
ASJC Scopus Sachgebiete
Schlagwörter
- Enantioselective biotransformation, Epoxidation, Flavoprotein, Fusion protein, Protein linker, Soil microorganism, Sulfoxidation, Two-component monooxygenase