Abstract
Two trans-o-hydroxybenzylidenepyruvate hydratase-aldolases named tHBP HA A and tHBP HA B were purified from a cell-free extract of naphthalenesulfonate-assimilating Sphingomonas paucimobilis (formerly Pseudomonas sp.) TA-2 to an electrophoretically homogeneous state by successive column chromatographies on DEAE-cellulose, DEAE-Toyopearl 650 M, Sephacryl S-100, Hydroxyapatite, and Mono Q. These enzymes were similar to each other in molecular mass (ca. 37 kDa on SDS-PAGE, ca. 110 kDa on ultracentrifugation), thermal stability (<50°C) and optimum pH (pH 9.0). However, they differed from each other in N-terminal amino acid sequences, pH stability, Km values for trans-o-hydroxybenzylidenepyruvate (tHBP), and inhibition by p-chloromercuribenzoic acid (PCMB). tHBP HA B had a homologous N-terminal amino acid sequence with tHBP HAs from Pseudomonas vesicularis DSM 6383 (strain BN 6) and Sphingomonas aromaticivorans F 119, and tHBP HA A had a homologous sequence with tHBP HAs of Pseudomonas putida strain OUS 82, Pseudomonas sp. strain C 18 and NAH 7 plasmid. tHBP HA B was inhibited by PCMB, but tHBP HA A was not. Their Km values for tHBP were 9 and 3 μM, respectively. tHBP HA B was stable in the range of pH 7.1 to pH 10.7, and tHBP HA A was stable in the range of pH 6.0 to 9.3.
