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Volume 148, Issue 8

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

Abstract

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 α subunit (BphA1) containing segments from sp. strain LB400 and P6, and of a hydroxylase β subunit (BphA2), a ferredoxin (BphA3) and a ferredoxin reductase (BphA4) from strain LB400. All hybrid 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 β subunit nor the N- and C-terminal regions of the α 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 α 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 or other host organisms.

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Keyword(s): aerobic PCB catabolism , ARHDO, aromatic-ring-hydroxylating dioxygenase , BDO, biphenyl dioxygenase , biphenyl dioxygenase , CB, chlorobiphenyl , chlorobiphenyls , heterologous gene expression , OE, overlap extension , PCB, polychlorobiphenyl and substrate-binding site
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2002-08-01
2024-04-20
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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 α subunit
Microbiology 148, 2439 (2002); https://doi.org/10.1099/00221287-148-8-2439
/content/journal/micro/10.1099/00221287-148-8-2439
/content/journal/micro/10.1099/00221287-148-8-2439
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