A thermostable beta-ketothiolase of polyhydroxyalkanoates (PHAs) in Thermus thermophilus: purification and biochemical properties.
Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as granules in the cytoplasm of the cells. The biosynthesis of PHAs, in the thermophilic bacterium T. thermophilus grown in a mineral medium supplemented with sodium gluconate as sole carbon source has been recently reported. Here, we report the purification at apparent homogeneity of a beta-ketoacyl-CoA thiolase from T. thermophilus, the first enzyme of the most common biosynthetic pathway for PHAs. B-Ketoacyl-CoA thiolase appeared as a single band of 45.5-kDa molecular mass on SDS/PAGE. The enzyme was purified 390-fold with 7% recovery. The native enzyme is a multimeric protein of a molecular mass of approximately of 182 kDa consisting of four identical subunits of 45.5 kDa, as identified by an in situ renaturation experiment on SDS-PAGE. The enzyme exhibited an optimal pH of approximately 8.0 and highest activity at 65 degrees C for both direction of the reaction. The thiolysis reaction showed a substrate inhibition at high concentrations; when one of the substrates (acetoacetyl CoA or CoA) is varied, while the concentrations of the second substrates (CoA or acetoacetyl CoA respectively) remain constant. The initial velocity kinetics showed a pattern of a family of parallel lines, which is in accordance with a ping-pong mechanism. beta-Ketothiolase had a relative low Km of 0.25 mM for acetyl-CoA and 11 microM and 25 microM for CoA and acetoacetyl-CoA, respectively. The enzyme was inhibited by treatment with 1 mM N-ethylmaleimide either in the presence or in the absence of 0.5 mM of acetyl-CoA suggesting that possibly a cysteine is located at/or near the active site of beta-ketothiolase.
Pantazaki
Anastasia A
AA
Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece.
Ioannou
Andrea K
AK
Kyriakidis
Dimitrios A
DA
D000102:Acetyl-CoA C-Acyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D011091:Polyesters / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D016963:Thermus thermophilus / Q000201:enzymology*
D004791:Enzyme Inhibitors; D011091:Polyesters; D021122:Protein Subunits; D000102:Acetyl-CoA C-Acyltransferase