CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION OF THE PLASMID-ENCODED GENES FOR THE 2-COMPONENT 2-HALOBENZOATE 1,2-DIOXYGENASE FROM PSEUDOMONAS-CEPACIA-2CBS

The two-component nonheme iron dioxygenase system 2-halobenzoate 1,2-d ioxygenase from Pseudomonas cepacia 2CBS catalyzes the double hydroxyl ation of 2-halobenzoates with concomitant release of halogenide and ca rbon dioxide, yielding catechol. The gene cluster encoding this enzyme , cbdABC, was localized on a 70-kbp conjugative plasmid designated pBA H1. The nucleotide sequences of cdbABC and flanking regions were deter mined. In the deduced amino acid sequence of the large subunit of the terminal oxygenase component (CbdA), a conserved motif proposed to bin d the Rieske-type [2Fe-2S] cluster was identified. In the NADH: accept or reductase component (CbdC), a putative binding site for a chloropla st-type [2Fe-2S] center and possible flavin adenine dinucleotide- and NAD-binding domains were identified. The cbdABC sequences show signifi cant homology to benABC, which encode benzoate 1,2-dioxygenase from Ac inetobacter calcoaceticus (52% identity at the deduced amino acid leve l), and to xylXYZ, which encode toluate 1,2-dioxygenase from Pseudomon as putida mt-2 (51% amino acid identity). Recombinant pKT231 harboring cbdABC and flanking regions complemented a plasmid-free mutant of wil d type P. cepacia 2CBS for growth on 2-chlorobenzoate, and it also all owed recombinant P. putida KT2440 to metabolize 2-chlorobenzoate. Func tional NADH:acceptor reductase and oxygenase components of 2-halobenzo ate 1,2-dioxygenase were enriched from recombinant Pseudomonas clones.