Isolation and Properties of Malic Enzyme and Its Gene in<i>Rhodopseudomonas palustris</i>No. 7
作者:Ikuo SATO、Jun YOSHIKAWA、Akiko FURUSAWA、Kazuhiro CHIKU、Seigo AMACHI、Takaaki FUJII
DOI:10.1271/bbb.90566
日期:2010.1.23
Malic enzyme (ME) was purified as an electrophoretically homogenous protein from Rhodopseudomonas palustris No. 7. The molecular weight of ME was estimated to be 650 kDa and that of its subunit, 86 kDa. ME activity was remarkably enhanced by di- and mono-valent cations, and the K a values for Mg2+ and NH4 + were 0.26 and 0.56 mm respectively. Purified ME used both NAD+ and NADP+ as electron acceptors, with K m values of 0.11 and 1.8 mm. The K m value for l-malate was 1.7 mm using NAD+ as electron acceptor. Gene cloning of the ME indicated that the ME from R. palustris strain No. 7 was composed of 774 amino acids encompassing the ME and phosphotransacetylase domains, although purified ME displayed no phosphotransacetylase activity. ME activity was inhibited by acetyl-CoA, oxaloacetate, and fructose-6-phosphate. These results suggest that ME plays an important role in the metabolic regulation of R. palustris No. 7 under photoheterotrophic conditions.
从嗜紫色非硫杆菌 No. 7 中纯化的苹果酸酶(ME)是一种电泳均一的蛋白质。ME 的分子量估计为 650 kDa,其亚基的分子量为 86 kDa。ME 活性受到二价和单价阳离子的显著增强,Mg2+ 和 NH4+ 的 K a 值分别为 0.26 和 0.56 mm。纯化的 ME 同时使用 NAD+ 和 NADP+ 作为电子受体,其 K m 值分别为 0.11 和 1.8 mm。使用 NAD+ 作为电子受体时,l-苹果酸的 K m 值为 1.7 mm。ME 的基因克隆显示,R. palustris No. 7 株的 ME 由 774 个氨基酸组成,涵盖了 ME 和磷酸转乙酰酶结构域,尽管纯化的 ME 并未显示出磷酸转乙酰酶活性。ME 活性被乙酰辅酶 A、草酰乙酸和果糖-6-磷酸抑制。这些结果表明,ME 在 R. palustris No. 7 的光异养条件下的代谢调控中发挥了重要作用。