An NADP-linked acetoacetyl CoA reductase from Zoogloea ramigera.
Zoogloea ramigera I-16 M was found to contain two stereospecific acetoacetyl CoA reductases; one was NADP+-linked and D(-)-beta-hydroxybutyryl CoA specific and the other was NAD+-linked and L(+)-isomer specific. The NADP+-linked enzyme, purified approximately 150-fold, had a pH optimum for the reduction of acetoacetyl CoA at 8.1, but no definite pH optimum for the oxidation for beta-hydroxybutyryl CoA. The apparent Michaelis constants for acetoacetyl CoA and NADPH were 8.3 and 21 micrometer, respectively. The enzyme was markedly inhibited by acetoacetyl CoA at concentrations higher than 10 micrometer. The incorporation of [1-14C]acetyl CoA into poly-beta-hydroxybutyrate (PHB) by bacterial crude extract (containing beta-ketothiolase, acetoacetyl CoA reductases, enoyl CoA hydratases and PHB synthases) or by a system reconstituted from purified preparations of beta-ketothiolase, acetoacetyl CoA reductase and PHB synthase, was observed only in the presence of NADPH, but not NADH. Among various enzymes involved in PHB metabolism, only the specific activity of glucose 6-phosphate dehydrogenase was elevated 5-fold within 2 h after the addition of glucose to the cells grown in the basal medium. These findings suggest that, in Z. ramigera I-16M, acetoacetyl CoA is directly reduced to D(-)-beta-hydroxybutyryl CoA by the NADP+-dependent reductase, and PHB synthesis is at least partially controled by NADPH availability through glucose 6-phosphate dehydrogenase.
D000090:Acetoacetates; D000105:Acetyl Coenzyme A / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D003065:Coenzyme A; D004746:Enoyl-CoA Hydratase / Q000494:pharmacology; D006885:Hydroxybutyrates / Q000096:biosynthesis; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D015045:Zoogloea / Q000201:enzymology*
D000090:Acetoacetates; D006885:Hydroxybutyrates; D009243:NAD; D009249:NADP; D000105:Acetyl Coenzyme A; D000429:Alcohol Oxidoreductases; D004746:Enoyl-CoA Hydratase; D003065:Coenzyme A