title: Extremely thermostable phosphoenolpyruvate carboxylase from an extreme thermophile, Rhodothermus obamensis.
abstract: Phosphoenolpyruvate carboxylase (PEPC) was purified from an extremely thermophilic bacterium, Rhodothermus obamensis, growing optimally at 80 degrees C, which had recently been isolated from a shallow marine hydrothermal vent in Japan. The native enzyme was a homotetramer of 400 kDa in molecular mass, as estimated by gel filtration chromatography, and the subunit exhibited an apparent molecular mass of 100 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum temperature for enzyme activity was 75 degrees C. The enzyme exhibited an absolute requirement for divalent cations and a pH optimum of 8.0. The enzyme was extremely thermostable and there was no loss of enzyme activity on incubation for 2 h at 85 degrees C. The enzyme exhibited a positive allosteric property with acetyl-CoA and fructose 1,6-bisphosphate, and a negative one with L-aspartate and L-malate. These effectors affected not only the thermophilicity but also the thermostability of the enzyme, and the substrate, co-factors, and salts increased the thermostability as well. The extrinsic thermostabilization might be a possible mechanism for adaptation of the enzyme to high temperature.
authors.affiliation: Department of Applied Bioscience, Graduate School of Agriculture, Kyoto University. kent@kais.kyoto-u.ac.jp
authors.forename: K
authors.initials: K
authors.lastname: Takai
authors.forename: Y
authors.initials: Y
authors.lastname: Sako
authors.forename: A
authors.initials: A
authors.lastname: Uchida
authors.forename: Y
authors.initials: Y
authors.lastname: Ishida
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology; D001224:Aspartic Acid / Q000378:metabolism / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010728:Phosphoenolpyruvate / Q000378:metabolism / Q000494:pharmacology; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature
chemical_list: D002412:Cations; D001224:Aspartic Acid; D000105:Acetyl Coenzyme A; D010728:Phosphoenolpyruvate; D010730:Phosphoenolpyruvate Carboxylase