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.

The widespread global distribution of chlorinated hydrocarbons, their high lipophilicity and their recalcitrance have contributed to their importance as environmental toxicants. Their metabolism under oxic and anoxic conditions mediated by prokaryotic and eukaryotic cells is discussed. Various aerobic bacteria are able to use chlorinated hydrocarbons as the sole source of carbon and energy and some anaerobic bacteria can use some of these as an artificial electron acceptor in reductive dechlorination. Liver enzymes are responsible for the formation of hydrophilic metabolites ready for excretion which often lead to highly reactive and potentially toxic intermediates. Whereas fungi, especially ligninolytic ones, usually only exhibit ‘side activities’ for chlorinated hydrocarbons. The capabilities of bacteria had led to the development of various bioremediation processes. Both, successes and failures within these processes are known. Therefore, current research aims at a better understanding of global community interactions. Key Concepts: Chlorinated hydrocarbons have been produced by the chemical industry since nearly a decade in large amounts, but they can also be observed as natural compounds, sometimes exceeding the industrially produced amounts. Even though toxicological properties have pushed the chlorochemistry into the focus of considerable debate and governmental regulatory action, chlorinated hydrocarbons remain essential for certain applications. The aerobic metabolism of chlorinated hydrocarbons by bacteria has been studied in detail and an immense amount of information is available on pathways, enzymes and genes involved in the mineralisation of chloroaromatics. The anaerobic degradation of chlorinated hydrocarbons is due to the capablity of anaerobic bacteria to use them in anaerobic respiration, which results in dechlorination. Dehalococcoides organisms are the most versatile reductive dehalogenators as being capable to dehalogenate chlorinated dioxins, biphenyls, benzenes and vinyl chloride, among others. Microbial activities have been widespread used for bioremediation purposes through natural attenuation, biostimulation and bioaugmentation. The poor understanding of the functioning of the complex microbial activities in situ made bioremediation efforts quite unreliable. The rapid development of molecular techniques in recent years allows immense insights into the processes in situ , but also on the overall physiology of biocatalysts.

No abstract