CHARACTERIZATION OF THE GENE ENCODING CATECHOL 2,3-DIOXYGENASE OF ALCALIGENES SP KF711 - OVEREXPRESSION, ENZYME-PURIFICATION, AND NUCLEOTIDE SEQUENCING

Catechol 2,3-dioxygenase (C23O) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of catechol to form 2-hydroxymucon ic semialdehyde, The C23O gene of Alcaligenes sp. KF711 was overexpres sed in Escherichia coli HB101 by using the lac promoter of pUC18, and its gene product was purified by using immunoaffinity chromatography. The purified C23O exhibited a 35-kDa single band on an SDS-polyacrylam ide gel, and its ring-fission activity on dihydroxylated aromatics was 4-methylcatechol > 4-chlorocatechhol > catechol > 3-methylcatechol mu ch greater than 2,3-dihydroxybiphenyl. Nucleotide sequence analysis of the C23O gene revealed an open reading frame of 927 bp, which can enc ode 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-po lyacrylamide gel. The amino acid sequence of the C23O was compared wit h those of nine other extradiol-type dioxygenases, including 2,3-dihyd roxybiphenyl dioxygenase (2,3-DHBD) and 1,2-dihydroxynaphthalene dioxy genase (1,8-DHND). The C23O of Alcaligenes sp. KF711 exhibited 80 to 9 4% identity in amino acid sequence with other C23Os, and 20 to 25% ide ntity with 1,2-DHND and 2,3-DHBDs, Furthermore, sequence comparison of 10 extradiol-type dioxygenases has led to identifying 19 evolutionari ly conserved amino acid residues whose possible catalytic roles are pr oposed. (C) 1996 Academic Press, Inc.