Abstract
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.
Original language | English (US) |
---|---|
Pages (from-to) | 1015-1024 |
Number of pages | 10 |
Journal | ChemBioChem |
Volume | 10 |
Issue number | 6 |
DOIs | |
State | Published - Apr 17 2009 |
Externally published | Yes |
Keywords
- Chloroaromatic degradation
- Cleavage reactions
- Metalloenzymes
- Oxidoreductases
- Protein engineering
ASJC Scopus subject areas
- Biochemistry
- Molecular Medicine
- Molecular Biology
- Organic Chemistry