Genetic and biochemical characterization of the broad spectrum chlorobenzene dioxygenase from Burkholderia sp. strain PS12 dechlorination of 1,2,4,5-tetrachlorobenzene

Abstract

The bacterium, Burkholderia (previously Pseudomonas) sp. strain PSI2, reported earlier to degrade 1,2,4-trichlorobenzene is shown here to utilize also 1,2,4,5-tetrachlorobenzene (Cl1-benzene) as a growth substrate. To investigate the possibility that this organism attacks Cl1-benzene with a chlorobenzenc dioxygenase which concomitantly causes dehalogenation, and to analyze the substrate range of the initial enzyme, a 5503-bp DNA fragment from PS12, exhibiting high similarity to genes coding for class IIB dioxygenases, was cloned and expressed in Escherichia coli. The sequence includes the tec genes coding for the α-subunit and β-subunit of a terminal dioxygenase, a ferredoxin and a reductase. E. coli cells producing these proteins were able to dioxygenolytically attack a range of aromatic compounds including chlorinated benzenes and toluene, and also dinuclear aromatics such as biphenyl and dibenzo-p-dioxin. The enzyme was shown by 18O2 incorporation experiments to dioxygenolytically attack a chlorosubstituted carbon atom of Cl4-benzene, thereby forming an unstable diol intermediate which spontaneously rearomatizes with concomitant chloride elimination to the corresponding 3,4,6-trichlorocatechol (Cl4-catechol).