Structure of catechol 2,3-dioxygenase gene from Alcaligenes eutrophus 335.
Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cleaving aromatic C-C bond at meta position 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 of Alcaligenes eutrophus 335, a strain degrading phenol and p-cresol, was cloned. The C23O gene was localized in an 1.4-kb PstI fragment from A. eutrophus 335, and was expressed in E. coli HB101. 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-dihydroxy-phenylacetate > 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 from A. eutrophus 335 exhibited the highest 59% identity with those of corresponding enzymes from Pseudomonas sp. CF600 (p VI150), P. putida HS1 (pDK1), and P. putida PpG7 (NAH7). An alignment of amino acid sequences of extradiol-type dioxygenases including C23O from A. eutrophus 335 has revealed that catalytically and structurally important amino acid residues of the enzymes were conserved during evolution.
Kang
B S
BS
College of Pharmacy, Chungbuk National University, Cheongju, Korea.
Ha
J Y
JY
Lim
J C
JC
Lee
J
J
Kim
C K
CK
Min
K R
KR
Kim
Y
Y
D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D050557:Catechol 2,3-Dioxygenase; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D049308:Dioxygenases; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010105:Oxygenases / Q000235:genetics*; D016415:Sequence Alignment
D004269:DNA, Bacterial; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase