Overexpression and biochemical characterization of soluble pyridine nucleotide transhydrogenase from Escherichia coli
Overexpression and biochemical characterization of soluble pyridine nucleotide transhydrogenase from Escherichia coli
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DOI：
10.1111/j.1574-6968.2011.02287.x
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发表时间：
2011-07-01
影响因子：
2.1
通讯作者：
Zhu, Guoping
中科院分区：
文献类型：
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作者：
Cao, Zhengyu;Song, Ping;Zhu, Guoping
可溶性吡啶核苷酸转氢酶 (STH) 是一种能量依赖性黄素蛋白，可直接催化 NAD(H) 和 NADP(H) 之间的氢化物转移，以维持这两种氧化还原辅因子的稳态。大肠杆菌中的 sth 基因被克隆并表达为融合蛋白（EcSTH）。纯化的 EcSTH 在 35 摄氏度、pH 7.5 时显示出最大活性。热灭活研究表明，50 摄氏度下 5 小时后，EcSTH 仍保留 50% 的活性。该酶在 4 摄氏度下可稳定保持 25 天。 EcSTH 的表观 K(m) 值对于 NADPH 为 68.29 μM，对于硫代-NAD+ 为 133.2 μM。 k(cat)/K(m) 比率表明，EcSTH 对 NADPH 的偏好是 thio-NAD+ 的 1.25 倍。产物抑制研究表明，EcSTH 活性会被过量的 NADPH 强烈抑制，但不会被硫代 NAD+ 抑制。 EcSTH 活性被 2 mM 腺嘌呤核苷酸增强，并被二价金属离子：Mn2+、Co2+、Zn2+、Ni2+ 和 Cu2+ 抑制。然而，预孵育30 min后，除Zn2+、Ni2+和Cu2+外，大多数二价金属离子对EcSTH活性几乎没有影响。酶分析可为EcSTH的利用提供重要的基础知识。
The soluble pyridine nucleotide transhydrogenase (STH) is an energy-independent flavoprotein that directly catalyzes hydride transfer between NAD(H) and NADP(H) to maintain homeostasis of these two redox cofactors. The sth gene in Escherichia coli was cloned and expressed as a fused protein (EcSTH). The purified EcSTH displayed maximal activity at 35 degrees C, pH 7.5. Heat-inactivation studies showed that EcSTH retains 50% activity after 5 h at 50 degrees C. The enzyme was stable at 4 degrees C for 25 days. The apparent K(m) values of EcSTH were 68.29 mu M for NADPH and 133.2 mu M for thio-NAD+. The k(cat)/K(m) ratios showed that EcSTH had a 1.25-fold preference for NADPH over thio-NAD+. Product inhibition studies showed that EcSTH activity was strongly inhibited by excess NADPH, but not by thio-NAD+. EcSTH activity was enhanced by 2 mM adenine nucleotide and inhibited by divalent metal ions: Mn2+, Co2+, Zn2+, Ni2+ and Cu2+. However, after preincubation for 30 min, most divalent metal ions had little effect on EcSTH activity, except Zn2+, Ni2+ and Cu2+. The enzymatic analysis could provide the important basic knowledge for EcSTH utilizations.