CHARACTERIZATION OF THE GENE ENCODING CATECHOL 2,3-DIOXYGENASE FROM ACHROMOBACTER-XYLOSOXIDANS KF701

Catechol 2,3-dioxygenase (C23O) catalyzes a meta cleavage of the aroma tic ring in catechol to form 2-hydroxymuconic semialdehyde. A C23O gen e was cloned from chromosomal DNA of A. xylosoxidans KF701, a soil bac terium degrading biphenyl, and expressed in E. coli HB101. In substrat e specificity to catechol and its analogs, the C23O exhibited the high est aromatic ring-fission activity to catechol, and its relative activ ity to other dihydroxylated aromatics was 4-chlorocatechol > 4-methylc atechol > 3-methylcatechol much greater than 2,3-dihydroxybiphenyl. Ar omatic ring-fission activity of the C23O to catechol was about 40-fold higher than that to 2,3-dihydroxybiphenyl. Nucleotide sequence analys is of the C23O gene from A. xylosoxidans KF701 revealed an open readin g frame consisting of 924 base pairs, and identified a putative riboso me-binding sequence (AGGTGA) at about 10 nucleotides upstream from the initiation codon. The open reading frame can encode a polypeptide cha in with molecular weight of 34 kDa containing 307 amino acid residues. The deduced amino acid sequence of the C23O exhibited the highest hom ology with that of C23O from Pseudomonas sp. IC with 96% identity, and the least homology with that of C23O from P. putida Fl with 22% ident ity among reported C23O sequences. Furthermore, comparison of the C23O sequence with other extradiol dioxygenases has led to identification of evolutionally conserved amino acid residues whose possible catalyti c and structural roles are proposed. (C) 1997 Academic Press.