1. Academic Validation
  2. Fine-tuning of catalytic properties of catechol 1,2-dioxygenase by active site tailoring

Fine-tuning of catalytic properties of catechol 1,2-dioxygenase by active site tailoring

  • Chembiochem. 2009 Apr 17;10(6):1015-24. doi: 10.1002/cbic.200800836.
Raffaella Caglio 1 Francesca Valetti Patrizia Caposio Giorgio Gribaudo Enrica Pessione Carlo Giunta
Affiliations

Affiliation

  • 1 Department of Human and Animal Biology, University of Torino, Italy.
Abstract

Catechol 1,2-dioxygenases and chlorocatechol dioxygenases are Fe(III)-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 K(M) 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 k(cat) towards chlorinated substrates. The biphasic Arrhenius plot observed in A72S suggests the involvement of a dynamic switch in the fine regulation of the Enzyme.

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