title: Directed evolution of a non-heme-iron-dependent extradiol catechol dioxygenase: identification of mutants with intradiol oxidative cleavage activity.
abstract: The non-heme-iron(II)-dependent extradiol catechol dioxygenases catalyse the oxidative cleavage of substituted catechols found on bacterial aromatic degradation pathways. The reaction mechanism of the extradiol dioxygenases is believed to proceed through the same proximal hydroperoxide intermediate as the iron(III)-dependent intradiol catechol dioxygenases. Directed evolution was carried out on members of the class III extradiol catechol dioxygenases, by using 1) error-prone polymerase chain reaction, 2) a primer-based cross-over method; the mutant dioxygenases were then screened for their ability to process a range of substituted catechols. Several mutant enzymes were found to show higher activity towards certain substituted catechols, including 4-chlorocatechol, and higher affinity for the iron(II) cofactor. Two mutants isolated from error-prone PCR of Escherichia coli MhpB (mutants R215W and K273R) were found to produce a mixture of extradiol and intradiol cleavage products, as detected by GC-MS and 1H NMR spectroscopy. The residue corresponding to K273 in protocatechuate 4,5-dioxygenase (LigAB), Val244, is located approximately 12 A from the iron(II) centre, but close to the putative dioxygen channel; R215 is found on a sequence loop not present in LigB.
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK.
authors.forename: Janne
authors.initials: J
authors.lastname: Schlosrich
authors.forename: Kirstin L
authors.initials: KL
authors.lastname: Eley
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Crowley
authors.forename: Timothy D H
authors.initials: TD
authors.lastname: Bugg
mesh_terms: D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015394:Molecular Structure; D016296:Mutagenesis; D009154:Mutation; D019159:Nonheme Iron Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D020578:Sphingomonas / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D019159:Nonheme Iron Proteins; D010105:Oxygenases; D049308:Dioxygenases; C083582:3-carboxyethylcatechol 2,3-dioxygenase; C022476:protocatechuate 4,5-dioxygenase; C439587:extradiol dioxygenase