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The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase subunit

German Research Centre for Biotechnology (GBF), Division of Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany¹

Author for correspondence: Bernd Hofer. Tel: +49 531 6181467. Fax: +49 531 6181411. e-mail: bho{at}gbf.de

Protein engineering by segment exchange was used to distinguish between regions of major and minor influence on the structure of the substrate-binding pocket of a biphenyl dioxygenase (BDO). Eight chimaeric enzyme systems were generated that each consisted of a hybrid hydroxylase subunit (BphA1) containing segments from Burkholderia sp. strain LB400 and Rhodococcus globerulus P6, and of a hydroxylase ß subunit (BphA2), a ferredoxin (BphA3) and a ferredoxin reductase (BphA4) from strain LB400. All hybrid bphA1 genes were expressed at high levels. Seven of the resulting fusion subunits functionally interacted with the other polypeptides of the dioxygenase system to yield catalytically active enzymes. Changes in the regiospecificity of substrate attack, monitored by the formation of seventeen different dioxygenation products obtained from seven chlorobiphenyls, were used to monitor effects of segment exchanges on the structure of the BDO substrate-binding site. Exchanges of neither the ß subunit nor the N- and C-terminal regions of the subunit exerted significant influences. All BDO regions that showed major effects on the substrate-binding pocket were located between approximately positions 165 and 395 of the subunit. Within this part of the enzyme, in addition to segments identified previously, a subregion which is involved in ligation of the mononuclear iron significantly influenced the regiospecificity of substrate dioxygenation. Moreover, the results indicate that the construction of appropriate hybrid genes may be used as a general strategy to overcome problems in obtaining heterologous BDO activities in Escherichia coli or other host organisms.

Keywords: aerobic PCB catabolism, biphenyl dioxygenase, chlorobiphenyls, heterologous gene expression, substrate-binding site

Abbreviations: ARHDO, aromatic-ring-hydroxylating dioxygenase; BDO, biphenyl dioxygenase; CB, chlorobiphenyl; OE, overlap extension; PCB, polychlorobiphenyl

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