STRUCTURE OF CATECHOL 2,3-DIOXYGENASE GENE FROM ALCALIGENES-EUTROPHUS-335

Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cl eaving aromatic C-C bond at meta position of dihydroxylated aromatic s ubstrates, catalyzes the conversion of catechol to 2-hydroxymuconic se mialdehyde. As our ongoing study to characterize biochemical and genet ic properties of the extradiol-type dioxygenases at molecular level, a C23O gene encoded in chromosomal DNA of Alcaligenes eutrophus 335, a strain degrading phenol and p-cresol, was cloned. The C23O gene was lo calized in an 1.4-kb PstI fragment from A. eutrophus 335, and was expr essed in E. coli HB101. The C23O exhibited the highest aromatic ring-f ission activity to catechol as a substrate, and its relative activity to other dihydroxylated aromatic substrates was in order of catechol > > 4-methylcatechol > 3-methylcatechhol, protocatechuate, 4-chlorocatec hol > 3,4-dihydroxyphenylacetate > 2,3-dihydrorrybiphenyl. Nucleotide sequence of the 1.4-kb fragment has revealed that an open reading fram e (ORF) corresponding to the C23O gene was composed of 930 base pairs. A putative ribosome-binding sequence of AGGAG was found at about 10 n ucleotides upstream the ORF which can encode a polypeptide of molecula r weight 34 kDa consisting of 309 amino acid residues. The deduced ami no acid sequence of C23O from A. eutrophus 335 exhibited the highest 5 9% identity with those of corresponding enzymes from Pseudomonas sp. C F600 (pVI150), P. putida HS1 (pDK1), and P. putida PpG7 (NAH7). An ali gnment of amino acid sequences of extradiol-type dioxygenases includin g C23O from A. eutrophus 335 has revealed that catalytically and struc turally important amino acid residues of the enzymes were conserved du ring evolution. (C) 1998 Academic Press.