A unique homodimeric NAD+-linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri
A unique homodimeric NAD+-linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri
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来自最小的自养真核生物金牛骨球菌的独特同型二聚体 NAD( ) 连接的异柠檬酸脱氢酶。
DOI：
10.1096/fj.14-257014
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发表时间：
2015-06-01
期刊：
影响因子：
4.8
通讯作者：
Zhu, Guo-Ping
中科院分区：
文献类型：
--
作者：
Tang, Wang-Gang;Song, Ping;Zhu, Guo-Ping
在真核生物中，依赖NAD(+)的异柠檬酸脱氢酶(IDH)是严格意义上的线粒体，是Krebs循环中的关键酶。到目前为止，所有已知的线粒体中的NAD(+)特异性IDH(NAD-IDH)都被认为是溶液中的异构体。在这里，一个独特的同源二聚体NAD-IDH从最小的自养性微真核生物-牛链球菌(OtIDH)中被揭开了面纱。活性OtIDH分子量约为93 kDa，亚基大小为46.7 kDa。在Mn2+和Mg2+存在下，OtIDH对NAD(+)的选择性分别是NADP(+)的42倍和51倍。有趣的是，与其他同二聚体同系物不同，OtIDH对异柠檬酸表现出S型动力学行为；与其他异低聚同系物不同，OtIDH对异柠檬酸(S-0.5和lt；10muM)具有极高的亲和力。此外，在进化轨迹的基础上，通过合理的突变，可以将其辅酶专一性从NAD(+)(古老性状)完全转化为NADP(+)(适应性性状)。突变体D344R和D344R/M345H对NADP(+)的偏好分别是NAD(+)的15倍和72倍，表明D344和M345是NAD(+)的决定因素。这些发现还表明，OtIDH可能是II型IDH的祖先形式(所有报道的成员都是NADP(+)连接的酶)，并可能进化为NADP(+)依赖的IDH，以适应碳饥饿下NADPH需求的增加。
In eukaryotes, NAD(+)-dependent isocitrate dehydrogenase (IDH) is strictly mitochondrial and is a key enzyme in the Krebs cycle. To date, all known NAD(+)-specific IDHs (NAD-IDHs) in the mitochondria are believed to be heteromeric in solution. Here, a unique homodimeric NAD-IDH from Ostreococcus tauri (OtIDH), the smallest autotrophic picoeukaryote, was unveiled. Active OtIDH has a molecular weight of similar to 93 kDa with each subunit of 46.7 kDa. In the presence of Mn2+ and Mg2+, OtIDH displayed 42-fold and 51-fold preference for NAD(+) over NADP(+), respectively. Interestingly, OtIDH exhibited a sigmoidal kinetic behavior in response to isocitrate unlike other homodimeric homologs, and a remarkably high affinity for isocitrate (S-0.5 < 10 mu M) unlike other heterooligomeric homologs. Furthermore, its coenzyme specificity can be completely converted from NAD(+) (ancient trait) to NADP(+) (adaptive trait) by rational mutagenesis based on the evolutionary trace. Mutants D344R and D344R/M345H displayed a 15-fold and 72-fold preference for NADP(+) over NAD(+), respectively, indicating that D344 and M345 are the determinants of NAD(+) specificity. These findings also suggest that OtIDH may be an ancestral form of type II IDHs (all reported members are NADP(+)-linked enzymes) and may have evolved into NADP(+)-dependent IDH for adaptation to the increased demand of NADPH under carbon starvation.