Investigation of acid-base catalysis in the extradiol and intradiol catechol dioxygenase reactions using a broad specificity mutant enzyme and model chemistry.

The extradiol and intradiol catechol dioxygenase reaction mechanisms proceed via a common proximal hydroperoxide intermediate, which is processed via different Criegee 1,2-rearrangements. An R215W mutant of extradiol dioxygenase MhpB, able to produce a mixture of extradiol and intradiol cleavage products, was analysed at pH 5.2-8.6, and the yield of extradiol product was found to be highly pH-dependent, whereas the yield of intradiol product was pH-independent. The acid-base chemistry of a biomimetic reaction for extradiol oxidative catechol cleavage was also investigated, using 1,4,7-triazacyclononane, FeCl(2), and pyridine in methanol, in which pyridine is proposed to act as both a general base and (in protonated form) a general acid. Kinetic experiments using a range of meta- and para-substituted pyridines gave a Brønsted plot of log(v) vs. pK(a) showing a bell-shaped plot. Oxidative catechol cleavage by a pyridine-monosubstituted beta-cyclodextrin in the presence of TACN and FeCl(2) in methanol yielded only intradiol cleavage products. It is therefore proposed that bifunctional acid-base catalysis is required for iron (ii)-dependent extradiol catechol cleavage, whereas the rate-determining step for intradiol catechol cleavage does not involve acid-base catalysis.

Directed evolution of a non-heme-iron-dependent extradiol catechol dioxygenase: identification of mutants with intradiol oxidative cleavage activity.

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.