Cloning of a gene encoding hydroxyquinol 1,2-dioxygenase that catalyzes both intradiol and extradiol ring cleavage of catechol

Two Escherichia coli transformants with catechol 1,2-dioxygenase activity w ere selected from a gene library of the benzamide-assimilating bacterium Ar throbacter species strain BA-5-17, which produces four catechol 1,2-dioxyge nase isozymes, A DNA fragment isolated from one transformant contained a co mplete open reading frame (ORF). The deduced amino acid sequence of the ORF shared high identity with hydroxyquinol 1,2-dioxygenase. An enzyme express ed by the ORF was purified to homogeneity and characterized. When hydroxyqu inol was used as a substrate, the purified enzyme showed 6.8-fold activity of that for catechol. On the basis of the sequence identity and substrate s pecificity of the enzyme, we concluded that the ORF encoded hydroxyquinol 1 ,2-dioxygenase. When catechol was used as a substrate, cis,cis-muconic acid and 2-hydroxymuconic 6-semialdehyde, which were products by the intradiol and extradiol ring cleavage activities, respectively, were produced. These results showed that the hydroxyquinol 1,2-dioxygenase reported here was a n ovel dioxygenase that catalyzed both the intradiol and extradiol cleavage o f catechol.