Competition between beta-ketothiolase and citrate synthase during poly(beta-hydroxybutyrate) synthesis in Methylobacterium rhodesianum.
The enzymes beta-ketothiolase and citrate synthase from the facultatively methylotrophic Methylobacterium rhodesianum MB 126, which uses the serine pathway, were purified and characterized. The beta-ketothiolase had a relatively high Km for acetyl-CoA (0.5 mM) and was strongly inhibited by CoA (Ki 0.02 mM). The citrate synthase had a much higher affinity for acetyl-CoA (Km 0.07 mM) and was significantly inhibited by NADH (Ki 0.15 mM). The intracellular concentration of CoA metabolites and nucleotides was determined in M. rhodesianum MB 126 during growth on methanol. The level of CoA decreased from about 0.6 nmol (mg dry mass)-1 during growth to the detection limit when poly(beta-hydroxybutyrate) (PHB) accumulated. Nearly unchanged intracellular concentrations of NADH, NADPH, and acetyl-CoA of about 0.5, 0.6-0.7, and 1.0 nmol (mg dry mass)-1, respectively, were determined during growth and PHB synthesis. During growth, the beta-ketothiolase was almost completely inhibited by CoA, and acetyl-CoA was principally consumed by the citrate synthase. During PHB accumulation, the beta-ketothiolase had about 75% of its maximum activity and showed much higher activity than citrate synthase, which at the actual NADH concentration was about 75% inhibited. NADPH concentration was sufficiently high to allow the unlimited activity of acetoacetyl-CoA reductase (Km NADPH 18 microM). PHB synthesis is probably mainly controlled by the CoA concentration in M. rhodesianum MB 126.
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Abteilung Biotechnologie, Fakultät für Biowissenschaften, Pharmazie und Psychologie, Universität Leipzig, Permoserstrasse 15, D-04318 Leipzig, Germany.
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D020155:3-Hydroxybutyric Acid; D000105:Acetyl Coenzyme A / Q000378:metabolism; D000102:Acetyl-CoA C-Acyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003065:Coenzyme A / Q000494:pharmacology; D003470:Culture Media / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism*; D007700:Kinetics; D000432:Methanol / Q000378:metabolism; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D011108:Polymers / Q000378:metabolism
D003470:Culture Media; D006885:Hydroxybutyrates; D011108:Polymers; D009243:NAD; D009249:NADP; D000105:Acetyl Coenzyme A; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase; D000102:Acetyl-CoA C-Acyltransferase; D002950:Citrate (si)-Synthase; D003065:Coenzyme A; D020155:3-Hydroxybutyric Acid; D000432:Methanol