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

Raffaella Caglio, Francesca Valetti, Patrizia Caposio, Giorgio Gribaudo, Enrica Pessione, Carlo Giunta

Research output: Contribution to journalArticle

19 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)1015-1024
Number of pages10
JournalChemBioChem
Volume10
Issue number6
DOIs
StatePublished - Apr 17 2009
Externally publishedYes

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Catechol 1,2-Dioxygenase
Catalytic Domain
Tuning
Enzymes
Protein Engineering
Arrhenius plots
Mutagenesis
Reducing Agents
Substrates
Site-Directed Mutagenesis
Catalysis
Kinetic parameters
Substitution reactions
Switches
Derivatives
Mutation
Proteins
catechol oxygenase II

Keywords

  • Chloroaromatic degradation
  • Cleavage reactions
  • Metalloenzymes
  • Oxidoreductases
  • Protein engineering

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry
  • Molecular Medicine
  • Molecular Biology

Cite this

Fine-tuning of catalytic properties of catechol 1,2-dioxygenase by active site tailoring. / Caglio, Raffaella; Valetti, Francesca; Caposio, Patrizia; Gribaudo, Giorgio; Pessione, Enrica; Giunta, Carlo.

In: ChemBioChem, Vol. 10, No. 6, 17.04.2009, p. 1015-1024.

Research output: Contribution to journalArticle

Caglio, R, Valetti, F, Caposio, P, Gribaudo, G, Pessione, E & Giunta, C 2009, 'Fine-tuning of catalytic properties of catechol 1,2-dioxygenase by active site tailoring', ChemBioChem, vol. 10, no. 6, pp. 1015-1024. https://doi.org/10.1002/cbic.200800836
Caglio, Raffaella ; Valetti, Francesca ; Caposio, Patrizia ; Gribaudo, Giorgio ; Pessione, Enrica ; Giunta, Carlo. / Fine-tuning of catalytic properties of catechol 1,2-dioxygenase by active site tailoring. In: ChemBioChem. 2009 ; Vol. 10, No. 6. pp. 1015-1024.
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