Shuto H, Fukui T, Saito T, Shirakura Y, Tomita K
Eur J Biochem. 1981 Aug;118(1):53-9. doi: 10.1111/j.1432-1033.1981.tb05485.x.
An NAD-linked acetoacetyl-CoA reductase of Zoolgoea 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 [Saito, T. et al (1977) Arch. Microbiol. 114, 211 - 217], the purified enzyme was strictly stereospecific to L(+)-3-hydroxybutyryl-CoA, and was active not only with NAD+ but also 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 microM and 6.5 microM, respectively, and in the oxidation reaction, Km values for L(+)-3-hydroxybutyryl-CoA and DNA+ were 7.0 microM and 32 microM, respectively. Among various 3-hydroxyacyl-CoAs 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. These findings indicate that the NAD-linked acetoacetyl-CoA reductase is not directly involved in the biosynthesis of poly(3-hydroxybutyrate).
对食油假单胞菌I-16-M的一种与NAD相关的乙酰乙酰辅酶A还原酶进行了纯化，使其达到电泳纯。与来自同一细菌的D(-)-3-羟基丁酰辅酶A特异性的与NADP相关的乙酰乙酰辅酶A还原酶[斋藤，T.等人(1977年)《微生物学文献》114，211 - 217]不同，纯化后的酶对L(+)-3-羟基丁酰辅酶A具有严格的立体特异性，并且不仅对NAD+有活性，对NADP+也有活性，尽管NADP+作为辅酶的效果不如NAD+。该酶在还原乙酰乙酰辅酶A时的最适pH为6.3，在氧化L(+)-3-羟基丁酰辅酶A时的最适pH为8.0。在还原反应中，乙酰乙酰辅酶A和NADH的Km值分别为8.8微摩尔和6.5微摩尔，在氧化反应中，L(+)-3-羟基丁酰辅酶A和NAD+的Km值分别为7.0微摩尔和32微摩尔。在测试的各种3-羟基酰基辅酶A中，L(+)-3-羟基丁酰辅酶A和L(+)-3-羟基戊酰辅酶A是最具活性的底物。当使用与NADP相关而非与NAD相关的还原酶时，观察到由纯化的3-氧硫解酶、乙酰乙酰辅酶A还原酶和聚(3-羟基丁酸)合酶制剂重构的系统从乙酰辅酶A合成聚(3-羟基丁酸)。这些发现表明，与NAD相关的乙酰乙酰辅酶A还原酶不直接参与聚(3-羟基丁酸)的生物合成。
