Cloning and Sequence Analysis of Hydroxyquinol 1,2-Dioxygenase Gene in 2,4,6-Trichlorophenol-Degrading Ralstonia pickettii DTP0602 and Characterization of Its Product
Hatta. T Kiyohara. H Nakano. O Imai. N Takizawa. N
Journal of bioscience and Bioengineering ; 1999 [Vol.87] Pages:267-272
Abstract
A gene encoding hydroxyquinol 1,Zdioxygenase was cloned from 2,4,6-trichlorophenol-degrading Ralstonia (Pseudomonas) pickettii strain DTP0602. Cell-free extracts of Escherichia coli containing a cloned 1.4-kb &I-XhoI DNA fragment of R. pickettii DTP0602 hydroxyquinol 1, Zdioxygenase converted hydroxyquinol into maleylacetate and also degraded 6-chlorohydroxyquinol. The 1.4-kb DNA fragment contained one open reading frame (designated hadO composed 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 puri6ed HadC protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of HadC exhibited high homology to that of the hydroxyquinol 1,Zdioxygenase of 2,4,5trichlorophenoxyacetic acid-degrading Burkholderfa cepacia AC1100 (Dauharas, D.L.et al., Appl. Environ. Microbial., 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 catalyzed the oxidation of pyrogallol and 3-methylcatechol, possible intermediates in the degradation of 2,4,6+ichlorophenol, in addition to 6-chlorohydroxyquinol and hydroxyquinol. The dioxygenase catalyzed both ortho- and meta-cleavage of 3-methylcatechol.
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