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Journal of Bacteriology, August 1999, p. 4805-4811, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Heterologous Expression and Characterization of the Purified Oxygenase Component of Rhodococcus globerulus P6 Biphenyl Dioxygenase and of Chimeras Derived from It

Hervé Chebrou,dagger Yves Hurtubise,Dagger Diane Barriault, and Michel Sylvestre*

INRS-Santé, Université du Québec, Pointe-Claire, Québec H9R 1G6, Canada

Received 10 March 1999/Accepted 21 May 1999

In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase. The alpha  or beta  subunit of P6 oxygenase was exchanged with the corresponding subunit of Pseudomonas sp. strain LB400 or of Comamonas testosteroni B-356 to create new chimeras that were purified ht-proteins and designated ht-alpha P6beta P6, ht-alpha P6beta LB400, ht-alpha P6beta B-356, ht-alpha LB400beta P6, and ht-alpha B-356beta P6. ht-alpha P6beta P6, ht-alpha P6beta LB400, ht-alpha P6beta B-356 were not expressed active in recombinant Escherichia coli cells carrying P6 bphA1 and bphA2, P6 bphA1 and LB400 bphE, or P6 bphA1 and B-356 bphE because the [2Fe-2S] Rieske cluster of P6 oxygenase alpha  subunit was not assembled correctly in these clones. On the other hand ht-alpha LB400beta P6 and ht-alpha B-356beta P6 were produced active in E. coli. Furthermore, active purified ht-alpha P6beta P6, ht-alpha P6beta LB400, ht-alpha P6beta B-356, showing typical spectra for Rieske-type proteins, were obtained from Pseudomonas putida KT2440 carrying constructions derived from the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce ht-proteins in Pseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an alpha  and a beta  subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.

* Corresponding author. Mailing address: INRS-Santé, 245 boul. Hymus, Pointe-Claire, Québec H9R 1G6, Canada. Phone: 514-630-8829. Fax: 514-630-8850. E-mail: michel.sylvestre{at}inrs-sante.uquebec.ca.

dagger Present address: Laboratoire de Biocatalyse, UST-Nantes, 44322 Nantes, France.

Dagger Present address: Laboratoires Choisy, Louiseville, Québec J5V 2L7, Canada.

Journal of Bacteriology, August 1999, p. 4805-4811, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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