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Journal of Bacteriology, August 2009, p. 4996-5009, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00307-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP{triangledown} ,{dagger}

Dirk Tischler,1* Dirk Eulberg,2,# Silvia Lakner,2 Stefan R. Kaschabek,1 Willem J. H. van Berkel,3 and Michael Schlömann1

Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09596 Freiberg, Germany,1 Department of Microbiology, University Stuttgart, Allmandring 31, 70569 Stuttgart, Germany,2 Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands3

Received 6 March 2009/ Accepted 21 May 2009

Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His10-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.

* Corresponding author. Mailing address: Environmental Microbiology, TU Bergakademie Freiberg, Leipziger Str. 29, 09596 Freiberg, Germany. Phone: 49-3731-393739. Fax: 49-3731-393012. E-mail: Dirk-Tischler{at}email.de

{triangledown} Published ahead of print on 29 May 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

# Present address: NOXXON Pharma AG, Max-Dohrn-Str. 8-10, D-10589 Berlin, Germany.

Journal of Bacteriology, August 2009, p. 4996-5009, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00307-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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