title: Fine-tuning of catalytic properties of catechol 1,2-dioxygenase by active site tailoring.
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.
authors.affiliation: Department of Human and Animal Biology, University of Torino, Italy.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Caglio
authors.forename: Francesca
authors.initials: F
authors.lastname: Valetti
authors.forename: Patrizia
authors.initials: P
authors.lastname: Caposio
authors.forename: Giorgio
authors.initials: G
authors.lastname: Gribaudo
authors.forename: Enrica
authors.initials: E
authors.lastname: Pessione
authors.forename: Carlo
authors.initials: C
authors.lastname: Giunta
mesh_terms: D000150:Acinetobacter / Q000201:enzymology; D000595:Amino Acid Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D050548:Catechol 1,2-Dioxygenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D050548:Catechol 1,2-Dioxygenase