CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION OF THE PLASMID-ENCODED GENES FOR THE 2-COMPONENT 2-HALOBENZOATE 1,2-DIOXYGENASE FROM PSEUDOMONAS-CEPACIA-2CBS
B. Haak et al., CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION OF THE PLASMID-ENCODED GENES FOR THE 2-COMPONENT 2-HALOBENZOATE 1,2-DIOXYGENASE FROM PSEUDOMONAS-CEPACIA-2CBS, Journal of bacteriology, 177(3), 1995, pp. 667-675
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.