Fine‐Tuning of Catalytic Properties of Catechol 1,2‐Dioxygenase by Active Site Tailoring

Choosing chloro: By reshaping the catalytic pocket of a catechol 1,2‐dioxygenase through a structural route alternative to evolution, novel engineered chlorocatechol dioxygenase‐like enzymes were obtained. Variants show an inversion of specificity with a preference for 4‐chlorocatechol and activity on the rarely recognised substrate 4,5‐dichlorocatechol. magnified image Catechol 1,2‐dioxygenases and chlorocatechol dioxygenases are FeIII‐dependent enzymes that do not require a reductant to perform the ortho cleavage of the aromatic ring. The reaction mechanism is common to the two enzymes, and active‐site residues must play a key role in the fine‐tuning of specificity. Protein engineering was applied for the first time to the catalytic pocket of a catechol 1,2‐dioxygenase by site‐specific and site‐saturation mutagenesis with the purpose of redesigning the pocket shape for improved catalysis on bulky derivatives. Mutants were analysed for changes in kinetic parameters: 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. A L69A substitution conveys gain‐of‐function activity towards 4‐tert‐butylcatechol. Mutations of position 72 enhance kcat towards chlorinated substrates. The biphasic Arrhenius plot observed in A72S suggests the involvement of a dynamic switch in the fine regulation of the enzyme.