Main conclusion

Hydroxy(phenyl)pyruvic acid reductase from Actaea racemosa catalyzes dual reactions in reducing 4-hydroxyphenylpyruvic acid as well as β-hydroxypyruvic acid. It thus qualifies to be part of fukinolic and cimicifugic acid biosynthesis and also photorespiration.

Abstract

The accumulation of fukinolic acid and cimicifugic acids is mainly restricted to Actaea racemosa (Ranunculaceae) and other species of the genus Actaea/Cimicifuga. Cimicifugic and fukinolic acids are composed of a hydroxycinnamic acid part esterified with a benzyltartaric acid moiety. The biosynthesis of the latter is unclear. We isolated cDNA encoding a hydroxy(phenyl)pyruvic acid reductase (GenBank OR393286) from suspension-cultured material of A. racemosa (ArH(P)PR) and expressed it in E. coli for protein production. The heterologously synthesized enzyme had a mass of 36.51 kDa and catalyzed the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid or β-hydroxypyruvic acid to glyceric acid, respectively. The optimal temperature was at 38 °C and the pH optimum at pH 7.5. NADPH is the preferred cosubstrate (K_m 23 ± 4 µM). Several substrates are accepted by ArH(P)PR with β-hydroxypyruvic acid (K_m 0.26 ± 0.12 mM) followed by 4-hydroxyphenylpyruvic acid (K_m 1.13 ± 0.12 mM) as the best ones. Thus, ArH(P)PR has properties of β-hydroxypyruvic acid reductase (involved in photorespiration) as well as hydroxyphenylpyruvic acid reductase (possibly involved in benzyltartaric acid formation).

中文翻译：

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总状Actaea racemosa L.中的羟基（苯基）丙酮酸还原酶：升麻酸和烟酸生物合成中的推定酶

主要结论

来自Actaea racemosa 的羟基(苯基)丙酮酸还原酶催化还原4-羟基苯基丙酮酸以及β-羟基丙酮酸的双重反应。因此，它有资格成为烟酸和升麻酸生物合成以及光呼吸的一部分。

抽象的

岩藻酸和升麻酸的积累主要限于总状毛茛科和升麻属的其他物种。升麻酸和岩藻酸由用苄基酒石酸部分酯化的羟基肉桂酸部分组成。后者的生物合成尚不清楚。我们从总状曲霉(ArH(P)PR)的悬浮培养材料中分离出编码羟基(苯基)丙酮酸还原酶 (GenBank OR393286) 的 cDNA，并将其在大肠杆菌中表达以产生蛋白质。异源合成的酶的质量为36.51 kDa，并分别催化4-羟基苯基丙酮酸向4-羟基苯基乳酸或β-羟基丙酮酸向甘油酸的NAD(P)H依赖性还原。最适温度为 38 °C，最适 pH 为 7.5。 NADPH 是首选的共底物 (K _m 23 ± 4 µM)。 ArH(P)PR 接受多种底物，其中 β-羟基丙酮酸 (K _m 0.26 ± 0.12 mM)，其次是 4-羟基苯基丙酮酸 (K _m 1.13 ± 0.12 mM)。因此，ArH(P)PR 具有 β-羟基丙酮酸还原酶（参与光呼吸）和羟苯基丙酮酸还原酶（可能参与苯甲基酒石酸形成）的特性。