Purification and Some Properties of 2-Hydroxychromene-2-Carboxylate Isomerase from Naphthalenesulfonate-Assimilating Pseudomonas sp. TA-2

Abstract

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 naphtha-lene 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 0.15mM.