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Journal of Bacteriology, December 2003, p. 6976-6980, Vol. 185, No. 23
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.23.6976-6980.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Pinpointing Biphenyl Dioxygenase Residues That Are Crucial for Substrate Interaction

Marco Zielinski,1 Silke Kahl,1 Hans-Jürgen Hecht,2 and Bernd Hofer1*

Division of Microbiology,1 Department of Structural Biology, German Research Centre for Biotechnology, D-38124 Braunschweig, Germany2

Received 21 April 2003/ Accepted 8 August 2003

Three regions of the biphenyl dioxygenase (BDO) of Burkholderia sp. strain LB400 have previously been shown to significantly influence the interaction between enzyme and substrates at the active site. For a further discrimination within these regions, we investigated the effects of 23 individual amino acid exchanges. The regiospecificity of substrate dioxygenation was used as a sensitive means to monitor changes in the steric-electronic structure of the active site. Replacements of residues that, according to a model of the BDO three-dimensional structure, directly interact with substrates in most, but not all, cases (Met231, Phe378, and Phe384) very strongly altered this parameter (by factors of >7). On the other hand, a number of amino acids (Ile243, Ile326, Phe332, Pro334, and Trp392) which have no contacts with substrates also strongly changed the site preference of dioxygenation (by factors of between 2.6 and 3.5). This demonstrates that residues which had not been predicted to be influential can play a pivotal role in BDO specificity.

* Corresponding author. Mailing address: GBF, Division of Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. Phone: (49-531) 6181467. Fax: (49-531) 6181411. E-mail: bho{at}gbf.de.

Journal of Bacteriology, December 2003, p. 6976-6980, Vol. 185, No. 23
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.23.6976-6980.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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