Characterization of the Gene Encoding Catechol 2,3-Dioxygenase ofAlcaligenessp. KF711: Overexpression, Enzyme Purification, and Nucleotide Sequencing

Abstract

Catechol 2,3-dioxygenase (C23O) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of catechol to form 2-hydroxymuconic semialdehyde. The C23O gene ofAlcaligenessp. KF711 was overexpressed inEscherichia coliHB101 by using thelacpromoter of pUC18, and its gene product was purified by using immunoaffinity chromatography. The purified C23O exhibited a 35-kDa single band on an SDS–polyacrylamide gel, and its ring-fission activity on dihydroxylated aromatics was 4-methylcatechol > 4-chlorocatechol > catechol > 3-methylcatechol << 2,3-dihydroxybiphenyl. Nucleotide sequence analysis of the C23O gene revealed an open reading frame of 927 bp, which can encode a polypeptide of 308 amino acid residues. The predicted molecular mass of 35 kDa is in agreement with that of purified C23O on an SDS–polyacrylamide gel. The amino acid sequence of the C23O was compared with those of nine other extradiol-type dioxygenases, including 2,3-dihydroxybiphenyl dioxygenase (2,3-DHBD) and 1,2-dihydroxynaphthalene dioxygenase (1,2-DHND). The C23O ofAlcaligenessp. KF711 exhibited 80 to 94% identity in amino acid sequence with other C23Os, and 20 to 25% identity with 1,2-DHND and 2,3-DHBDs. Furthermore, sequence comparison of 10 extradiol-type dioxygenases has led to identifying 19 evolutionarily conserved amino acid residues whose possible catalytic roles are proposed.