Purification and characterization of NADP-linked acetoacetyl-CoA reductase from Zoogloea ramigera I-16-M.
An NADP-linked acetoacetyl-CoA reductase was purified to electrophoretic homogeneity from Zoogloea ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium. The purified enzyme showed specific activity of 412 mumol acetoacetyl-CoA reduced per min per mg protein, which constituted an 880-fold purification compared to the crude extract, with a 32% yield. Electrophoretic analysis of the purified enzyme which had been cross-linked with dimethylsuberimidate showed that the native enzyme (Mr 92,000) is a tetramer of four identical subunits (Mr 25,500). Among the various D-(-)- and L-(+)-3-hydroxyacyl-CoAs tested, the purified enzyme oxidized only D-(-)-3-hydroxybutyryl-CoA and to a lesser extent D-(-)-3-hydroxyvaleryl-CoA in the presence of NADP+. The antiserum prepared against the purified enzyme completely inhibited poly(3-hydroxybutyrate) synthesis from acetyl-CoA by a crude extract of Z. ramigera I-16-M cells. These findings indicate that this enzyme plays an indispensable role as the supplier of D-(-)-3-hydroxybutyryl-CoA in poly(3-hydroxybutyrate) synthesis in this bacterium.
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D000907:Antibodies, Bacterial / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006885:Hydroxybutyrates / Q000096:biosynthesis; D008970:Molecular Weight; D009249:NADP / Q000302:isolation & purification*; D011091:Polyesters; D011108:Polymers / Q000096:biosynthesis; D013379:Substrate Specificity; D015045:Zoogloea / Q000201:enzymology*; D000094122:Polyhydroxybutyrates
D000429:Alcohol Oxidoreductases; D000907:Antibodies, Bacterial; D006885:Hydroxybutyrates; D009249:NADP; D011091:Polyesters; D011108:Polymers; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C023744:acetoacetyl-CoA reductase