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J. Biol. Chem., Vol. 279, Issue 46, 47480-47488, November 12, 2004

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Evolution of the Biphenyl Dioxygenase BphA from Burkholderia xenovorans LB400 by Random Mutagenesis of Multiple Sites in Region III^*

Diane Barriault and Michel Sylvestre

From the Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, 245 Boulevard Hymus, Pointe-Claire, Québec H9R 1G6, Canada

It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) subunit are involved in substrate recognition and regiospecificity toward chlorobiphenyls. However, the sequence pattern of the amino acids of that segment of seven amino acids located in the C-terminal portion of the subunit is rather limited in BPDOs of natural occurrence. In this work, we have randomly mutated simultaneously four residues (Thr³³⁵-Phe³³⁶-Ile³³⁸-Ile³⁴¹) of region III of Burkholderia xenovorans LB400 BphA. The library was screened for variants able to oxygenate 2,2'-dichlorobiphenyl (2,2'-CB). Replacement of Phe³³⁶ with Met or Ile with a concomitant change of Thr³³⁵ to Ala created new variants that transformed 2,2'-CB into 3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl, which is a dead end metabolite that was not cleaved by BphC. Replacement of Thr³³⁵-Phe³³⁶ with Ala³³⁵-Leu³³⁶ did not cause this type of phenotypic change. Regiospecificity toward congeners other than 2,2'-CB that were oxygenated more efficiently by variant Ala³³⁵-Met³³⁶ than by LB400 BPDO was similar for both enzymes. Thus structural changes that altered the regiospecificity toward 2,2'-CB did not affect the metabolite profile of other congeners, although it affected the rate of conversion of these congeners. It was especially noteworthy that both LB400 BPDO and the Ala³³⁵-Met³³⁶ variant generated 2,3-dihydroxy-2',4,4'-trichlorobiphenyl as the sole metabolite from 2,4,2',4'-CB and 4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl as the major metabolite from 2,3,2',3'-CB. This shows that 2,4,2',4'-CB is oxygenated principally onto vicinal ortho-meta carbons 2 and 3 and that 2,3,2',3'-CB is oxygenated onto meta-para carbons 4 and 5 by both enzymes. The data suggest that interactions between the chlorine substitutes on the phenyl ring and specific amino acid residues of the protein influence the orientation of the phenyl ring inside the catalytic pocket.

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

^* This work was supported by Natural Sciences and Engineering Research Council Grants 224153 and 257566. 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. Tel.: 514-630-8829; Fax: 514-630-8850; E-mail: Michel.Sylvestre{at}inrs-iaf.uquebec.ca.

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