Extremely thermostable phosphoenolpyruvate carboxylase from an extreme thermophile, Rhodothermus obamensis.
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.
Takai
K
K
Department of Applied Bioscience, Graduate School of Agriculture, Kyoto University. kent@kais.kyoto-u.ac.jp
Sako
Y
Y
Uchida
A
A
Ishida
Y
Y
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
D002412:Cations; D001224:Aspartic Acid; D000105:Acetyl Coenzyme A; D010728:Phosphoenolpyruvate; D010730:Phosphoenolpyruvate Carboxylase