Competition between β-ketothiolase and citrate synthase during poly(β-hydroxybutyrate) synthesis inMethylobacterium rhodesianum

Abstract

The enzymes β-ketothiolase and citrate synthase from the facultatively methylotrophicMethylobacterium rhodesianum MB 126, which uses the serine pathway, were purified and characterized. The β-ketothiolase had a relatively highK _m for acetyl-CoA (0.5 mM) and was strongly inhibited by CoA (K _i 0.02 mM). The citrate synthase had a much higher affinity for acetyl-CoA (K _m 0.07 mM) and was significantly inhibited by NADH (K _i 0.15 mM). The intracellular concentration of CoA metabolites and nucleotides was determined inM. 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(β-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 β-ketothiolase was almost completely inhibited by CoA, and acetyl-CoA was principally consumed by the citrate synthase. During PHB accumulation, the β-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 (K _mNADPH 18 μM). PHB synthesis is probably mainly controlled by the CoA concentration inM. rhodesianum MB 126.