Cloning and sequence analysis of hydroxyquinol 1,2-dioxygenase gene in 2,4,6-trichlorophenol-degrading Ralstonia pickettii DTP0602 and characterization of its product

A gene encoding hydroxyquinol 1,2-dioxygenase was cloned from 2,4,6-trichlo rophenol-degrading Ralstonia (Pseudomonas) pickettii strain DTP0602. Cell-f ree extracts of Escherichia coli containing a cloned 1.4-kb StuI-XhoI DNA f ragment of R. pickettii DTP0602 hydroxyquinol 1,2-dioxygenase converted hyd roxyquinol into maleylacetate and also degraded 6-chlorohydroxyquinol. The 1.4-kb DNA fragment contained one open reading frame (designated hadC) comp osed of 948 nucleotides. The molecular mass of 34,591 deduced from the gene product (HadC) was in agreement with the size (35 kDa) of the purified Had C protein determined by sodium dodecyl sulfate-polyacrylamide gel electroph oresis. The amino acid sequence of HadC exhibited high homology to that of the hydroxyqninol 1,2-dioxygenase of 2,4,5-trichlorophenoxyacetic acid-degr ading Burkholderia cepacia AC1100 (Daubaras, D. L. et al,, Appl. Environ. M icrobiol., 61, 1279-1289, 1995). The active enzyme had a molecular mass of 68 kDa, suggesting that it is functional as a homodimer. The enzyme also ca talyzed the oxidation of pyrogallol and 3-methylcatechol, possible intermed iates in the degradation of 2, 4,6-trichlorophenol, in addition to 6-chloro hydroxyquinol and hydroxyquinol. The dioxygenase catalyzed both ortho- and meta-cleavage of 3-methylcatechol.