Purification and some properties of 2-hydroxychromene-2-carboxylate isomerase from naphthalenesulfonate-assimilating Pseudomonas sp. TA-2.
A 2-hydroxychromene-2-carboxylate isomerase was purified from a cell-free extract of naphthalenesulfonate-assimilating Pseudomonas sp. TA-2 to an electrophoretically homogeneous state by successive column chromatography on DEAE-cellulose, DEAE-Toyopearl 650M, Sephadex G-75, Hydroxyapatite, and Mono Q. The enzyme had a molecular mass of 25 and 27 kDa as estimated by SDS-PAGE and Superdex 200, respectively. Its N-terminal 30 amino acid sequence had high homology with the deduced amino acid sequences of the 2HC2CA isomerase of nahD (a gene of naphthalene metabolism), pahD (a gene of naphthalene and phenanthrene metabolism), and doxJ (a gene of dibenzothiophene metabolism). The enzymatic product was a trans isomer. The isomerase activity was inhibited in the presence of monoiodoacetate or Hg2+, but not by preincubation with monoiodoacetate or N-ethylmaleimide. GSH functioned as a cofactor and activated the enzyme at above 0.15 mM.
The Osaka Municipal Technical Research Institute, Joto-ku, Osaka, 536-8553, Japan. ohmoto@omtri.city.osaka.jp
T
T
Ohmoto
T
T
Kinoshita
K
K
Moriyoshi
K
K
Sakai
N
N
Hamada
T
T
Ohe
D000595:Amino Acid Sequence; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009282:Naphthalenesulfonates / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D009282:Naphthalenesulfonates; D019746:Intramolecular Oxidoreductases; C078381:2-hydroxychromene-2-carboxylate isomerase