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

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 Fe2+. 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 a-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 ringhydroxylating oxygenases. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.