GENETIC AND BIOCHEMICAL-CHARACTERIZATION OF THE BROAD-SPECTRUM CHLOROBENZENE DIOXYGENASE FROM BURKHOLDERIA SP STRAIN PS12 - DECHLORINATION OF 1,2,4,5-TETRACHLOROBENZENE

The bacterium, Burkholderia (previously Pseudomonas) sp, strain PS12, reported earlier to degrade 1,2,4-trichlorobenzene is shown here to ut ilize also 1,2,4,5-tetrachlorobenzene (Cl-4-benzene) as a growth subst rate. To investigate the possibility that this organism attacks Cl-4-b enzene with a chlorobenzene dioxygenase which concomitantly causes deh alogenation, 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 Escher ichia coli. The sequence includes the tec genes coding for the alpha-s ubunit and beta-subunit of a terminal dioxygenase, a ferredoxin and a reductase. E. coli cells producing these proteins were able to dioxyge nolytically attack a range of aromatic compounds including chlorinated benzenes and toluene, and also dinuclear aromatics such as biphenyl a nd dibenzo-p-dioxin. The enzyme was shown by O-18(2) incorporation exp eriments to dioxygenolytically attack a chlorosubstituted carbon atom of Cl-4-benzene, thereby forming an unstable diol intermediate which s pontaneously rearomatizes with concomitant chloride elimination to the corresponding 3,4,6-trichlorocatechol (Cl-3-catechol).