Open AccessPurification and gene cloning of the oxygenase component of the terephthalate 1,2‐dioxygenase system from Delftia tsuruhatensis strain T7
Author(s) -
Shigematsu Toru,
Yumihara Kazuyo,
Ueda Yutaka,
Morimura Shigeru,
Kida Kenji
Publication year - 2003
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1016/s0378-1097(03)00124-1
Subject(s) - oxygenase , dioxygenase , protein subunit , peptide sequence , biochemistry , chemistry , amino acid , stereochemistry , biology , enzyme , gene
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 α 3 β 3 subunit structure was deduced from the molecular masses of 235, 46 and 17 kDa of the native complex and the α‐ and β‐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 α‐subunit was determined. The amino acid sequence of the α‐subunit (TerZα) 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 TerZα 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.