NADP+-Specific Isocitrate Dehydrogenase from Oleaginous Yeast Yarrowia lipolytica CLIB122: Biochemical Characterization and Coenzyme Sites Evaluation
NADP+-Specific Isocitrate Dehydrogenase from Oleaginous Yeast Yarrowia lipolytica CLIB122: Biochemical Characterization and Coenzyme Sites Evaluation
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DOI：
10.1007/s12010-013-0373-1
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发表时间：
2013-09-01
影响因子：
3
通讯作者：
Zhu, Guoping
中科院分区：
文献类型：
--
作者：
Li, Xue;Wang, Peng;Zhu, Guoping
脂解耶氏菌CLIB122 NADP（+）依赖性异柠檬酸脱氢酶（ylipp）被过表达和纯化。YlIDP的分子质量约为81.3 kDa，表明其在溶液中具有同二聚体结构。YlIDP是二价阳离子依赖性的，Mg2+被发现是最有利的辅助因子。纯化后的重组ylipp在55℃下表现出最大的催化活性，其最佳催化pH在8.5左右。热失活研究表明，重组YlIDP在45℃以下是稳定的，但在45℃以上其活性迅速下降。YlIDP完全依赖于NADP(+)，未检测到NADP依赖性活性。NADP（+）和异柠檬酸盐的K (m)值分别为59和31 μ m (Mg2+)， 120 μ m和58 μ m （Mn2+）。构建突变酶，初步改变YlIDP的辅酶特异性。R322D突变体NADP +的K (m)值为2410 μ m，是野生型酶的41倍左右。R322D突变体检测到NAD（+）依赖性活性，NAD（+）的K (m)和K （cat）值分别为47000 μ m和0.38 s（-1）。虽然R322D突变体对NAD（+）的活性较低，但它揭示了将真核IDP改造为NAD（+）依赖性IDP的可行性。
NADP(+)-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg2+ was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 A degrees C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 A degrees C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP(+) and no NAD-dependent activity could be detected. The K (m) values displayed for NADP(+) and isocitrate were 59 and 31 mu M (Mg2+), 120 mu M and 58 mu M (Mn2+), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K (m) values for NADP(+) of R322D mutant was 2,410 mu M, being about 41-fold higher than that of wild type enzyme. NAD(+)-dependent activity was detected for R322D mutant and the K (m) and k (cat) values for NAD(+) were 47,000 mu M and 0.38 s(-1), respectively. Although the R322D mutant showed low activity with NAD(+), it revealed the feasibility of engineering an eukaryotic IDP to a NAD(+)-dependent one.