Purification and gene cloning of the oxygenase component of the terephthalate 1,2-dioxygenase system from<i>Delftia tsuruhatensis</i>strain T7

Abstract

The terephthalate 1,2-dioxygenase system (TERDOS) was found in cell extracts of Delftia tsuruhatensis strain T7 (=IFO16741) grown in terephthalate-salt medium. The cell extract was separated by anion exchange chromatography to yield two fractions (R and Z) that were necessary for oxygenation of terephthalate with NADH and Fe(2+). The oxygenase component of TERDOS (TerZ) was purified from fraction Z by gel filtration chromatography to near homogeneity. An alpha(3)beta(3) subunit structure was deduced from the molecular masses of 235, 46 and 17 kDa of the native complex and the alpha- and beta-subunits, respectively. The N-terminal amino acid sequences of the two subunits of TerZ allowed polymerase chain reaction primers to be deduced and the DNA sequence of the alpha-subunit was determined. The amino acid sequence of the alpha-subunit (TerZalpha) showed significant similarities to the large subunits of multicomponent ring-hydroxylating oxygenases. Two motifs in the deduced amino acid sequence, a Rieske [2Fe-2S] center and a mononuclear Fe(II) binding site, were observed. Phylogenetic analyses indicated that TerZalpha and the large oxygenase component subunits ortho-halobenzoate 1,2-dioxygenase and salicylate-5-hydroxylase form a cluster that is distant from the rest of the large oxygenase subunits of multicomponent ring-hydroxylating oxygenases.