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Journal of Bacteriology, November 1998, p. 5828-5835, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Involvement of the Terminal Oxygenase beta  Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls

Yves Hurtubise, Diane Barriault, and Michel Sylvestre*

Institut National de la Recherche Scientifique---Santé, Pointe-Claire, Québec, H9R 1G6 Canada

Received 22 May 1998/Accepted 3 September 1998

Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 and in Pseudomonas sp. strain LB400. The enzyme comprises a two-subunit (alpha  and beta ) iron sulfur protein (ISPBPH), a ferredoxin (FERBPH), and a ferredoxin reductase (REDBPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3'-dichlorobiphenyl over the double-para-substituted congener 4,4'-dichlorobiphenyl or the double-ortho-substituted congener 2,2'-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2'-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2',5,5'-tetrachlorobiphenyl on adjacent meta-para carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISPBPH alpha  or beta  subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the alpha  subunit of ISPBPH of strain LB400 and the beta  subunit of ISPBPH of strain B-356 (the alpha LB400beta B-356 chimera) and the alpha B-356beta LB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISPBPH beta  subunit influences BPH dox's reactivity pattern toward chlorobiphenyls. Thus, if the alpha  subunit were the sole determinant of the enzyme reactivity pattern, the alpha B-356beta LB400 chimera should have behaved like B-356 ISPBPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISPBPH. On the other hand, the alpha LB400beta B-356 chimera showed features of both B-356 and LB400 ISPBPH where the enzyme was able to metabolize 2,2'- and 3,3'-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2',5,5'-tetrachlorobiphenyl.

* 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.

Journal of Bacteriology, November 1998, p. 5828-5835, Vol. 180, No. 22
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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