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

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 Mr of 150,000 (containing identical Mr 30,000 sub‐units) and the NADPH‐linked enzyme appeared to be a tetramer (Mr 84,000) with identical sub‐units (Mr 23,000). Kmapp 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.