IDENTIFICATION OF CHLOROBENZENE DIOXYGENASE SEQUENCE ELEMENTS INVOLVED IN DECHLORINATION OF 1,2,4,5-TETRACHLOROBENZENE

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate s pecificities. Escherichia coli tells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1 ,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene , A hybrid TecA dioxygenase variant containing the large alpha-subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity. Acquisition of dehalogenase activity was achieved by replacement of s pecific todC1 alpha-subunit subsequences by equivalent sequences of th e tecA1 alpha-subunit. Substrate transformation specificities and rate s by E. coli resting cells expressing hybrid systems were analyzed by high-performance liquid chromatography, This allowed the identificatio n of both a single amino acid and potentially interacting regions requ ired for dechlorination of tetrachlorobenzene, Hybrids with extended s ubstrate ranges were generated that exhibited activity toward both ben zene and tetrachlorobenzene. The regions determining substrate specifi city in (chloro)benzene dioxygenases appear to be different from those previously identified in biphenyl dioxygenases.