This publication is dedicated to the memory of Helmut Guth who pushed us to start enzyme modelling.
Degradation of chloroaromatics: structure and catalytic activities of wild-type chlorocatechol 2,3-dioxygenases and modified ones
Article first published online: 23 JUL 2012
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd
Special Issue: Baeza
Volume 15, Issue 1, pages 183–190, January 2013
How to Cite
Schmidt, E., Mandt, C., janssen, D. B., Pieper, D. H. and Reineke, W. (2013), Degradation of chloroaromatics: structure and catalytic activities of wild-type chlorocatechol 2,3-dioxygenases and modified ones. Environmental Microbiology, 15: 183–190. doi: 10.1111/j.1462-2920.2012.02831.x
This publication is dedicated to Kenneth N. Timmis, an important partner when starting research on microbial degradation of xenobiotic chemicals. Ken strongly pushed the topic environmental microbiology.
- Issue published online: 3 JAN 2013
- Article first published online: 23 JUL 2012
- Accepted manuscript online: 3 JUL 2012 01:01AM EST
- Received 29 March, 2012; revised 25 June, 2012; accepted 27 June, 2012.
To improve the efficiency and to investigate the molecular determinants that direct substrate specificity of chlorocatechol 2,3-dioxygenase CbzEGJ31, several mutant enzymes were constructed. Loci for substitutions of amino acids were selected by sequence comparisons as well as by homology modelling of known chlorocatechol 2,3-dioxygenases (CbzEBASF, CbzESK1 and CbzE16-6A). Activity measurements with various catechols showed that most of the modifications influenced activity only to a minor degree. The amino acid at position 154 seems to be located at a non-important position in the enzyme with minor extension into the substrate tunnel. Similarly, the change of related amino acids such as D95E and Y223F did not influence the catalysis since both residues are far away from the catalytic centre and the substrate tunnel. Even the modification of isoleucine to threonine in position 310, located at the outer substrate tunnel, showed a significant alteration of activities. Position 196 seems to be of higher relevance since the modification of valine to alanine, i.e. the reduction of the side-chain, produced much alteration. The amino acid is located at the interface of inner to outer substrate tunnel. CbzEV196A showed high relative kcat for 3-chlorocatechol. A pronounced increase in activity for 3-chlorocatechol resulted by the change from alanine to valine and from aspartic acid to glycine laying in the outer substrate tunnel at position 211 and 212 respectively.