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Journal of Bacteriology, August 2004, p. 5292-5302, Vol. 186, No. 16
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.16.5292-5302.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Biochemical Characterization of StyAB from Pseudomonas sp. Strain VLB120 as a Two-Component Flavin-Diffusible Monooxygenase

Katja Otto, Karin Hofstetter, Martina Röthlisberger, Bernard Witholt, and Andreas Schmid*

Institute of Biotechnology, ETHZ, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPT, CH-8093, Zurich, Switzerland

Received 10 March 2004/ Accepted 6 May 2004

Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of CC double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg–1 (kcat = 1.6 s–1) and Km values for styrene of 0.45 ± 0.05 mM (wild type) and 0.38 ± 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg–1 (kcat [NADH] = 60 s–1). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.

* Corresponding author. Mailing address: Institute of Biotechnology, ETHZ, Swiss Federal Institute of Technology, ETH Hoenggerberg, HPT, CH-8093, Zurich, Switzerland. Phone: 41 1 6333691. Fax: 41 1 6331051. E-mail: andreas.schmid{at}biotech.biol.ethz.ch.

Journal of Bacteriology, August 2004, p. 5292-5302, Vol. 186, No. 16
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.16.5292-5302.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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