Degradation of chloroaromatics: structure and catalytic activities of wild‐type chlorocatechol 2,3‐dioxygenases and modified ones

Abstract

Summary 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.

Factors affecting activity of catechol 2,3-dioxygenase from 2-chlorophenol-degradingStenotrophomonas maltophiliastrain KB2

“…Within this large group of enzymes catechol 2,3-dioxygenase (C23O) catalyzes ring cleavage of catechol and its chloro-, methyl-, and ethyl-derivatives (Bertini et al 1994;Okuta et al 2004;Schmidt et al 2012;Zhao et al 2011). The degradation process is affected by many environmental factors such as temperature, pH, pollutant concentration, presence of inhibitors, and stimulators as well as microbial competition and adaptation (Dokic et al 2011;Yeom & Yoo 1997;Schmidt et al 2012;Zhao et al 2011).…”

“…As the interaction of iron (II) with phenolate moieties of tyrosine residues plays an important role in the enzyme function (Ajao et al 2012;Palaniandavar & Mayilmurugan 2007;Wojcieszy ń ska et al 2011b;Schmidt et al 2012;Yeom & Yoo 1997), oxidation of this ion inactivates the enzyme (Hassett et al 2000).…”

Inactivation of 4-Oxalocrotonate Tautomerase by 5-Halo-2-hydroxy-2,4-pentadienoates

“…There are two well-characterized examples of "roadblocks" in meta-fission pathways (3,4). The first involves the mechanism-based inhibition of extradiol-type dioxygenases (e.g., catechol 2,3-dioxygenase) by halogenated catechols (2,4). It was initially thought that ring opening generated an acyl chloride species, which results in the covalent modification of the enzyme.…”

“…Halogenated aromatic compounds have great commercial, industrial, and pharmaceutical significance (1)(2)(3). Past and present uses include solvents, lubricants, dielectric fluids, insecticides, herbicides, flame retardants, drugs, and synthetic intermediates.…”

From the test tube to the environment – and back

“…But it happens that ( inter alia ) P. putida strain GJ31 grows on chlorobenzene using a meta pathway with chlorocatechol 2,3‐dioxygenase (CbzE) as a key step. This noteworthy enzyme has received since much attention and Schmidt and colleagues () explain their efforts for understanding the molecular determinants that cause substrate specificity of such CbzE product. Forward engineering of functional substrate binding sites in enzymes remains as one of the holy grails of industrial and environmental engineering and the last few years have witnessed both spectacular advances and not less spectacular failures in this endeavour.…”