Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680
Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680
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DOI：
10.1007/s11033-010-0486-3
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发表时间：
2011-08-01
影响因子：
2.8
通讯作者：
Zhu, Guo-Ping
中科院分区：
文献类型：
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作者：
Wang, Ao;Cao, Zheng-Yu;Zhu, Guo-Ping
来自多细胞原核生物除虫链霉菌 MA-4680 (SaIDH) 的单体 NADP 依赖性异柠檬酸脱氢酶在大肠杆菌中异质表达，并且 His 标记的酶进一步纯化至均质。 SaIDH 的分子量约为 80 kDa，这是单体异柠檬酸脱氢酶的典型分子量。基于结构的序列比对表明，推导的SaIDH氨基酸序列与已知的单体异柠檬酸脱氢酶表现出高度的序列同一性，并且辅酶、底物和金属离子结合位点完全保守。 SaIDH 的最佳 pH 值和温度分别为 pH 9.4 和 45A 摄氏度。热灭活研究表明，在 50A 摄氏度下加热 20 分钟会导致酶活性损失 50%。此外，SaIDH 对作为电子受体的 NADP(+) 具有绝对特异性。使用 Mn2+ 作为二价阳离子，NADP(+) 的表观 K (m) 值分别为 4.98 μM，NAD(+) 的表观 K (m) 值分别为 6,620 μM。该酶对 NADP(+) 的特异性 (k (cat)/K (m)) 比 NAD(+) 高 33,000 倍。此外，SaIDH 活性完全依赖于 Mn2+ 或 Mg2+ 的存在，但受到 Ca2+ 和 Zn2+ 的强烈抑制。综上所述，我们的研究结果表明重组 SaIDH 是一种二价阳离子依赖性单体异柠檬酸脱氢酶，其对 NADP(+) 具有非常高的辅因子偏好。
A monomeric NADP-dependent isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680 (SaIDH) was heteroexpressed in Escherichia coli, and the His-tagged enzyme was further purified to homogeneity. The molecular weight of SaIDH was about 80 kDa which is typical for monomeric isocitrate dehydrogenases. Structure-based sequence alignment reveals that the deduced amino acid sequence of SaIDH shows high sequence identity with known momomeric isocitrate dehydrogenase, and the coenzyme, substrate and metal ion binding sites are completely conserved. The optimal pH and temperature of SaIDH were found to be pH 9.4 and 45A degrees C, respectively. Heat-inactivation studies showed that heating for 20 min at 50A degrees C caused a 50% loss in enzymatic activity. In addition, SaIDH was absolutely specific for NADP(+) as electron acceptor. Apparent K (m) values were 4.98 mu M for NADP(+) and 6,620 mu M for NAD(+), respectively, using Mn2+ as divalent cation. The enzyme performed a 33,000-fold greater specificity (k (cat)/K (m)) for NADP(+) than NAD(+). Moreover, SaIDH activity was entirely dependent on the presence of Mn2+ or Mg2+, but was strongly inhibited by Ca2+ and Zn2+. Taken together, our findings implicate the recombinant SaIDH is a divalent cation-dependent monomeric isocitrate dehydrogenase which presents a remarkably high cofactor preference for NADP(+).