Suicide Inactivation of Catechol 2,3-Dioxygenase from Pseudomonas putida mt-2 by 3-Halocatechols

Abstract

The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation ( K 2 ) were 1.62 × 10 −3 sec −1 for 3-chlorocatechol and 2.38 × 10 −3 sec −1 for 3-fluorocatechol. The inhibitor constants ( K i ) were 23 μM for 3-chlorocatechol and 17 μM for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol, suggesting 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta -cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.

Microbial breakdown of halogenated aromatic pesticides and related compounds

“…The majority of organisms that degrade chlorocatechols do so via an ortho-cleavage pathway. meta-Cleavage of halocatechois by 2,3-dioxygenases was found to produce dead-end or suicide metabolites [31,32]. The acylchlorides act as acylating agents inactivating the ring cleavage enzyme irreversibly and therefore bacteria that utilize chlorocatechols usually express orthocleavage activity, meta-Cleavage of chlorocatechols has occasionally been observed.…”

Metabolism of 4-chlorophenol by Azotobacter sp. GP1: Structure of the meta cleavage product of 4-chlorocatechol

“…Whereas ortho cleavage has been shown to yield a productive pathway [7], meta cleavage may be lethal for the organism. Thus by meta cleavage of 3-chlorocatechol a highly reactive acyl chloride is formed leading to suicide inactivation of the meta cleavage enzyme [8].…”

Biodegradation of aromatic compounds by microalgae

“…The algal extracts also oxygenated 3-methylcatechol, 4-methylcatechol, 4-bromocatechol and 4-£uorocatechol to the corresponding ring cleavage products, which showed the expected shifts in spectral peaks under acid and alkaline conditions. Inhibition studies using 4-isopropylcatechol [40] and 3-chlorocatechol [37] gave results corresponding to those of bacterial studies, showing irreversible and suicide inhibitions, respectively. Speci¢c activities in these extracts for catechol 1,2-dioxygenase (catechol:oxygen 1,2-oxidoreductase; EC 1.13.11.1), the enzyme initiating the ortho pathway, were found to be negligible.…”

“…These activities were elucidated using whole cells with the alga having the highest speci¢city for phenol, then p-cresol, o-cresol and ¢nally m-cresol. Catabolism of phenol to catechol was con-¢rmed by the isolation of the product from incubations in which the catechol 2,3-dioxygenase (catechol:oxygen 2,3-oxidoreductase; EC 1.13.11.2) was inhibited with 3-chlorocatechol [26], which was shown earlier to suicide inhibit the enzyme [37].…”