Presence of two trans-o-hydroxybenzylidenepyruvate hydratase-aldolases in naphthalenesulfonate-assimilating Sphingomonas paucimobilis TA-2: comparison of some properties.
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 650M, 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 degrees C) and optimum pH (pH 9.0). However, they differed from each other in N-terminal amino acid sequences, pH stability, K(m) 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 BN6) and Sphingomonas aromaticivorans F119, and tHBP HA A had a homologous sequence with tHBP HAs of Pseudomonas putida strain OUS82, Pseudomonas sp. strain C18 and NAH7 plasmid. tHBP HA B was inhibited by PCMB, but tHBP HA A was not. Their K(m) values for tHBP were 9 and 3 M, 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.
Ohmoto
T
T
The Osaka Municipal Technical Research Institute, Morinomiya, Joto-ku, Osaka 536-8553, Japan. ohmoto@omtri.city.osaka.jp
Moriyoshi
K
K
Sakai
K
K
Hamada
N
N
Ohe
T
T
D000595:Amino Acid Sequence; D002474:Cell-Free System; D002614:Chelating Agents / Q000737:chemistry; D004795:Enzyme Stability; D006836:Hydro-Lyases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008673:Metals, Alkaline Earth / Q000737:chemistry; D019216:Metals, Heavy / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009282:Naphthalenesulfonates / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D020578:Sphingomonas / Q000201:enzymology* / Q000254:growth & development; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013696:Temperature
D002614:Chelating Agents; D008673:Metals, Alkaline Earth; D019216:Metals, Heavy; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D006836:Hydro-Lyases; C078380:trans-2-hydroxybenzylidenepyruvate hydratase-aldolase