An NAD‐Linked Acetoacetyl‐CoA Reductase from Zoogloea ramigera I‐16‐M

Abstract

An NAD-linked acetoacetyl-CoA reductase (EC 1.1.1.36) of Z. ramigera I-16-M was purified to electrophoretic homogeneity. In contrast to the D(-)-3-hydroxybutyryl-CoA-specific NADP-linked acetoacetyl-CoA reductase from the same bacterium the purified enzyme was strictly stereospecific to L(+)-3-hydroxybutyryl-CoA, and was active with NAD+ and with NADP+, although NADP+ was less effective than NAD+ as coenzyme. The enzyme showed a pH optimum at 6.3 for the reduction of acetoacetyl-CoA and at 8.0 for the oxidation of L(+)-3-hydroxybutyryl-CoA. In the reduction reaction, Km values for acetoacetyl-CoA and NADH were 8.8 and 6.5 .mu.M, respectively, and in the oxidation reaction, Km values for L(+)-3-hydroxybutyryl-CoA and NAD+ were 7.0 and 32 .mu.M, respectively. Among various 3-hydroxyacyl-CoA tested, L(+)-3-hydroxybutyryl-CoA and L(+)-3-hydroxyvaleryl-CoA were the most active substrates. Poly(3-hydroxybutyrate) synthesis from acetyl-CoA, by a system reconstituted from purified preparations of 3-oxothiolase, acetoacetyl-CoA reductase and poly(3-hydroxybutyrate) synthase, was observed when the NADP-linked but not the NAD-linked reductase was used. NAD-linked acetoacetyl-CoA reductase is apparently not directly involved in the biosynthesis of poly(3-hydroxybutyrate).