Cloning, DNA sequencing, and amino acid sequencing of catechol 1,2-dioxygenases (pyrocatechase) from Pseudomonas putida mt-2 and Pseudomonas arvilla C-1.
Catechol 1,2-dioxygenase catalyzes the oxygenative ring cleavage of catechol to form cis,cis-muconic acid and is encoded by a catA gene. We have cloned a catA gene from Pseudomonas putida mt-2 using a PCR product of amino acid sequence-based primers as a probe. The amino acid sequence deduced from the 930 nucleotides was in complete agreement with the chemically determined sequence of the protein. Crude extracts of Escherichia coli cells carrying the catA gene downstream from the lac promoter showed the enzyme activity. By using the same probe, we also cloned and sequenced the catA beta gene for catechol 1,2-dioxygenase isozyme beta beta from Pseudomonas arvilla C-1, which has three isozymes, alpha alpha, alpha beta, and beta beta (C. Nakai, H. Horiike, S. Kuramitsu, H. Kagamiyama, and M. Nozaki, 1990, J. Biol. Chem. 265, 660-665). There was very high homology between isozyme beta beta of the C-1 strain and the enzyme of the mt-2 strain in both the amino acid (98%) and the DNA sequences (97%). A preference for the use of codons terminating in C and G was found in the coding region of both the enzymes, which contributed to the high G + C content (65-66%) of the genes. A comparison of the DNA sequences of various catA genes from other sources revealed their common ancestry, whereas a comparison of the amino acid sequences of the enzymes revealed clear reflection of substrate specificity. Tyrosyl and histidyl residues for proposed ligands of ferric ion are conserved in all catechol 1,2-dioxygenases.
Nakai
C
C
Department of Biochemistry, Shiga University of Medical Science, Japan.
Uyeyama
H
H
Kagamiyama
H
H
Nakazawa
T
T
Inouye
S
S
Kishi
F
F
Nakazawa
A
A
Nozaki
M
M
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D050548:Catechol 1,2-Dioxygenase; D003001:Cloning, Molecular; D015342:DNA Probes; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D049308:Dioxygenases; D015723:Gene Library; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D015342:DNA Probes; D004269:DNA, Bacterial; D007527:Isoenzymes; D011994:Recombinant Proteins; D010105:Oxygenases; D049308:Dioxygenases; D050548:Catechol 1,2-Dioxygenase