CHANGE IN SUBSTRATE SPECIFICITY: TURNING A CATECHOL 1,2 DIOXYGENASE INTO A CHLOROCATECHOL 1,2 DIOXYGENASE

Oxygenases are key enzymes in degrading toxic compounds (pollutants, drugs and other xenobiotics) and therefore their potential application as biocatalysts is of great interest. Catechol 1,2 dioxygenases and chlorocatechol dioxygenases are FeIII-dependent enzymes involved in the detoxification cascade of aromatic compounds, since they catalyse the reaction that cleaves either the intradiol or the proximal extradiol bond of catechol, resulting in ring opening and thus de-aromatising the substrate. The reaction mechanism is common to the two enzymes and active site residues must play a key role in the fine tune of specificity. Protein engineering was applied for the first time to the catalytic pocket of a catechol 1,2 dioxygenase by site specific (position 69) and site saturation mutagenesis (position 72) with the purpose of redesigning the pocket shape for improved catalysis on bulky derivatives (1). The activity of the wt and mutants was assayed to determinate the kinetic parameters and the functional properties (pH, temperature, Ea measurement, demetallation) using the natural substrate, methylcatechols, chlorocatechols and other substituted diols, that are of environmental interest. Variants for residue 69 show an inversion of specificity with a preference towards 4-chlorocatechol (decrease of KM by a factor of 20) and activity on the rarely recognised substrate 4,5-dichlorocatechol, thus creating a novel engineered chlorocatechol dioxygenase. L69A substitution conveys gain-of-function-activity towards 4-tert-butylcatechol, a toxic compound found as contaminant in cosmetic preparations where the presence in trace is allowed by an European legislation (2). Mutations of position 72 (A72G, A72S, A72D, A72N, A72P) enhance kcat towards chlorinated substrates. A biphasic Arrhenius plot observed in A72S suggests the involvement of a dynamic switch in enzyme fine regulation (3). REFERENCES (1) R. Caglio, F. Valetti, P. Caposio, G. Gribaudo, E. Pessione, C. Giunta, ”Fine tuning of catalytic properties of catechol 1,2 dioxygenase by active site tailoring” ChemBioChem, 2009 (in press).