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J. Biol. Chem., Vol. 279, Issue 46, 47489-47496, November 12, 2004

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Revisiting the Regiospecificity of Burkholderia xenovorans LB400 Biphenyl Dioxygenase toward 2,2'-Dichlorobiphenyl and 2,3,2',3'-Tetrachlorobiphenyl^*

Diane Barriault, François Lépine, Mahmood Mohammadi, Sylvain Milot, Nicolas Leberre, and Michel Sylvestre

From the Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, Laval, Québec H7V 1B7, Canada

2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d₈ was determined to be a dihydroxychlorobiphenyl-d₇ by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.

Received for publication, June 17, 2004 , and in revised form, August 19, 2004.

^* This work was supported by National Science and Engineering Research Council of Canada grants 257566 and 0039579. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: INRS-IAF, 245 Boul Hymus, Pointe-Claire, Québec H9R 1G6, Canada. Tel.: 514-630-8829; Fax: 514-630-8850; E-mail: michel.sylvestre{at}inrs-iaf.uquebec.ca.

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