Purification and characterization of NADPH-dependent acetoacetyl-CoA reductase from Methylobacterium extorquens

Abstract

NADPH-dependent acetoacetyl-CoA (AcAc-CoA) reductase (EC 1.1.1.36) was purified to electrophoretic homogeneity from the methylotroph Methylobacterium extorquens – a producer of poly-3-hydroxybutyrate. The enzyme has an Mr of 141 000 and consists of four identical subunits (Mr 31 000) and demonstrates absolute specificity for NADPH as cofactor. NADPH-AcAc-CoA reductase is inhibited by NADPH, AcAc-CoA, NADP and NAD but is activated by isocitrate and ATP. The calculated Km values were 11.6 and 41 μM for AcAc-CoA and NADPH, respectively. The results suggest that this enzyme plays a more important role in the coordinated operation of the tricarboxylic acid cycle and poly-3-hydroxybutyrate synthesis in M. extorquens than in the taxonomically related M. rhodesianum MB 126.