COMPETITION BETWEEN BETA-KETOTHIOLASE AND CITRATE SYNTHASE DURING POLY(BETA-HYDROXYBUTYRATE) SYNTHESIS IN METHYLOBACTERIUM-RHODESIANUM

The enzymes beta-ketothiolase and citrate synthase from the facultativ ely methylotrophic Methylobacterium rhodesianum MB 126, which uses the serine pathway, were purified and characterized. The beta-ketothiolas e had a relatively high K-m for acetyl-CoA (0.5 mM) and was strongly i nhibited 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 metaboli tes and nucleotides was determined in M. rhodesianum MB 126 during gro wth on methanol. The level of CoA decreased from about 0.6 nmol (mg dr y mass)(-1) during growth to the detection limit when poly(beta-hydrox ybutyrate) (PHB) accumulated. Nearly unchanged intracellular concentra tions of NADH, NADPH, and acetyl-CoA of about 0.5, 0.6-0.7, and 1.0 nm ol (mg dry mass)(-1), respectively, were determined during growth and PHB synthesis. During growth, the beta-ketothiolase was almost complet ely inhibited by CoA, and acetyl-CoA was principally consumed by the c itrate synthase. During PHB accumulation, the beta-ketothiolase had ab out 75% of its maximum activity and showed much higher activity than c itrate synthase, which at the actual NADH concentration was about 75% inhibited. NADPH concentration was sufficiently high to allow the unli mited activity of acetoacetyl-CoA reductase (K-m NADPH 18 mu M). PHB s ynthesis is probably mainly controlled by the CoA concentration in M. rhodesianum MB 126.