The role of NADH- and NADPH-linked acetoacetyl-CoA reductases in the poly-3-hydroxybutyrate synthesizing organism Alcaligenes eutrophus

Abstract

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Alcaligenes eutrophus. Incorporation of [1-¹⁴C]-acetyl-CoA into poly-3-hydroxybutyrate (PHB) by systems reconstituted from purified preparations of either 3-ketothiolase, AcAc-CoA reductase and PHB synthase, occurred only when NADPH-AcAc-CoA reductase was present. The NADH reductase was active with all of the d(−)- and l(+)-3-hydroxyacyl-CoA substrates tested (C₄-C₁₀), whereas the NADPH reductase was only active with d(−)-3-hydroxyacyl-CoAs (C₄-C₆). The products of AcAc-CoA reduction by the NADH- and NADPH-linked enzymes were l(+)-3-hydroxybutyryl-CoA and d(−)-3-hydroxybutyryl-CoA, respectively. The NADH-linked enzyme had an M_r of 150,000 (containing identical M_r 30,000 sub-units) and the NADPH-linked enzyme appeared to be a tetramer (M_r 84,000) with identical sub-units (M_r 23,000). K_m^app values of 22 µM and 5 µM for AcAc-CoA and 13 µM (NADH) and 19 µM (NADPH) for the coenzymes were determined for the NADH- and NADPH-linked enzymes, respectively.