Structure of Catechol 2,3-Dioxygenase Gene fromAlcaligenes eutrophus335

Abstract

Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cleaving aromatic C-C bond atmetaposition of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. As our ongoing study to characterize biochemical and genetic properties of the extradiol-type dioxygenases at molecular level, a C23O gene encoded in chromosomal DNA ofAlcaligenes eutrophus335, a strain degrading phenol andp-cresol, was cloned. The C23O gene was localized in an 1.4-kbPstI fragment fromA. eutrophus335, and was expressed inE. coliHB101. The C23O exhibited the highest aromatic ring-fission activity to catechol as a substrate, and its relative activity to other dihydroxylated aromatic substrates was in order of catechol >> 4-methylcatechol > 3-methylcatechol, protocatechuate, 4-chlorocatechol > 3,4-dihydroxyphenylacetate > 2,3-dihydroxybiphenyl. Nucleotide sequence of the 1.4-kb fragment has revealed that an open reading frame (ORF) corresponding to the C23O gene was composed of 930 base pairs. A putative ribosome-binding sequence of AGGAG was found at about 10 nucleotides upstream the ORF which can encode a polypeptide of molecular weight 34 kDa consisting of 309 amino acid residues. The deduced amino acid sequence of C23O fromA. eutrophus335 exhibited the highest 59% identity with those of corresponding enzymes fromPseudomonassp. CF600 (pVI150),P. putidaHS1 (pDK1), andP. putidaPpG7 (NAH7). An alignment of amino acid sequences of extradiol-type dioxygenases including C23O fromA. eutrophus335 has revealed that catalytically and structurally important amino acid residues of the enzymes were conserved during evolution.