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Difference in kinetic behaviour of catechol 2,3-dioxygenase variants from a polluted environment

¹ Department of Environmental Microbiology, GBF – German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany
² Department of Structural Biology, GBF – German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany

Correspondence
Dietmar H. Pieper
dpi{at}gbf.de

In a previous environmental survey of a polluted area, the authors identified two catechol 2,3-dioxygenase (C23O) sequences predominant in environmental bacterial isolates mineralizing benzene and/or toluene and also in soil DNA extracts. In the present study, using information of stable operon arrangement and conserved gene sequences, the complete C23O ORFs of these two variants were cloned, sequenced and overexpressed. The variants differ in six nucleotide positions, and the putative protein sequences differ only by a single amino acid, Tyr or His, at position 218. Even though the three-dimensional model does not suggest a significant influence of such an amino acid substitution on enzyme function, the Tyr218 variant differed significantly from the His218 variant in lower turnover number and in lower apparent K_m for catecholic substrates. These results are evidence of the importance for enzyme function of amino acids not directly influencing active site structure and prove the utility of recovering polymorphisms evolved and selected for special functions in natural ecosystems.

The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this paper are AJ544924 and AJ544930.

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