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 Psudomonas 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 catAβ gene for catechol 1,2-dioxygenase isozyme ββ from Pseudomonas arvilla C-1, which has three isozymes, αα, αβ, and ββ (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 ββ 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.