PURIFICATION AND CHARACTERIZATION OF NADPH-DEPENDENT ACETOACETYL-COA REDUCTASE FROM METHYLOBACTERIUM-EXTORQUENS

NADPH-dependent acetoacetyl-CoA (AcAc-CoA) reductase (EC 1.1.1.36) was purified to electrophoretic homogeneity from the methylotroph Methylo bacterium extorquens - a producer of poly-3-hydroxybutyrate. The enzym e has an M-r of 141000 and consists of four identical subunits (M-r 31 000) and demonstrates absolute specificity for NADPH as cofactor. NADP H-AcAc-CoA reductase is inhibited by NADPH, AcAc-CoA, NADP and NAD but is activated by isocitrate and ATP. The calculated K-m values were 11 .6 and 41 mu M for AcAc-CoA and NADPH, respectively. The results sugge st that this enzyme plays a more important role in the coordinated ope ration of the tricarboxylic acid cycle and poly-3-hydroxybutyrate synt hesis in M. extorquens than in the taxonomically related M. rhodesianu m MB 126.