Mutagenesis of Sequence Determinants of Truncated Porcine ALOX15 Induces Changes in the Reaction Specificity by Altering the Catalytic Mechanism of Initial Hydrogen Abstraction.
Mutagenesis of Sequence Determinants of Truncated Porcine ALOX15 Induces Changes in the Reaction Specificity by Altering the Catalytic Mechanism of Initial Hydrogen Abstraction.
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截短猪 ALOX15 序列决定子的诱变通过改变初始夺氢的催化机制来诱导反应特异性的变化
DOI：
10.1002/chem.201704672
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
González-Lafont À
中科院分区：
文献类型：
--
作者：
Saura P;Kaganer I;Heydeck D;Lluch JM;Kühn H;González-Lafont À
脂氧合酶的反应特异性具有生理相关性，因为各种氧化产物表现出不同的生物活性。在哺乳动物ALOX 15直系同源物中，存在花生四烯酸12-和15-脂氧合酶，最近的研究表明，在灵长类动物发育后期，该反应特异性存在进化开关。先前的报告表明，12-脂氧合ALOX 15直系同源物可以通过某些序列决定簇的定点突变转化为15-脂氧合酶。不幸的是，这些改变的分子基础还没有得到很好的理解。在这里，猪ALOX 15的花生四烯酸12-脂氧合N-末端截短变体（其晶体结构可用）用于探索Val 418和Val 419序列决定簇诱变诱导的特异性转换的催化机制。我们发现Val 418 Ile + Val 419 Met双突变体主要是15-脂氧合。对接和MD模拟以及量子力学/分子力学计算表明，花生四烯酸15-脂氧合的野生型能垒比12-脂氧合高3.4 kcalmol-1。 相比之下，对于Val 418 Ile + Val 419 Met双突变体，12-脂氧合的能垒比15-脂氧合高6.0 kcal mol− 1。 我们的数据表明，酶-底物复合物的几何形状决定了这些能量屏障的值，因此，决定了ALOX 15同源物的反应特异性。
The reaction specificity of lipoxygenases is of physiological relevance since the various oxygenation products exhibit different biological activities. Among mammalian ALOX15 orthologs there are arachidonic acid 12‐ and 15‐lipoxygenating enzymes and recent studies suggested an evolutionary switch in that reaction specificity during late primate development. Previous reports showed that 12‐lipoxygenating ALOX15 orthologs can be converted to 15‐lipoxygenating enzymes by site‐directed mutagenesis of some sequence determinants. Unfortunately, the molecular basis for those alterations are not well understood. Here, the arachidonic acid 12‐lipoxygenating N‐terminal truncation variant of pig ALOX15, for which a crystal structure is available, was used to explore the catalytic mechanism of the specificity switch induced by mutagenesis of Val418 and Val419 sequence determinants. We found that Val418Ile+Val419Met double mutant is dominantly 15‐lipoxygenating. Docking and MD simulations, and quantum mechanics/molecular mechanics calculations indicated that the wildtype energy barrier for arachidonic acid 15‐lipoxygenation is 3.4 kcal mol−1higher than for 12‐lipoxygenation. In contrast, for the Val418Ile+Val419Met double mutant the energy barrier for 12‐lipoxygenation is 6.0 kcal mol−1higher than for 15‐lipoxygenation. Our data suggest that enzyme–substrate complex geometries determine the value of these energy barriers and, as a consequence, the reaction specificity of ALOX15 orthologs.
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DOI：
10.1016/j.bbalip.2014.10.002
发表时间：
2015-04
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Kuhn H;Banthiya S;van Leyen K
通讯作者：
van Leyen K
影响因子：
12.9
作者：
Patricia Saura;R. Suardíaz;Laura Masgrau;Àngels González-Lafont;E. Rosta;J. M. Lluch
通讯作者：
Patricia Saura;R. Suardíaz;Laura Masgrau;Àngels González-Lafont;E. Rosta;J. M. Lluch
DOI：
10.1016/0005-2760(94)90234-8
发表时间：
1994-01
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
H. Suzuki;K. Kishimoto;T. Yoshimoto;S. Yamamoto;F. Kanai;Y. Ebina;A. Miyatake;T. Tanabe
通讯作者：
H. Suzuki;K. Kishimoto;T. Yoshimoto;S. Yamamoto;F. Kanai;Y. Ebina;A. Miyatake;T. Tanabe
影响因子：
2.9
作者：
Jacquot, Cyril;Wecksler, Aaron T.;van der Donk, Wilfred A.
通讯作者：
van der Donk, Wilfred A.
了解人类酶 15-脂氧合酶-2 催化的花生四烯酸夺氢机制。
影响因子：
5.5
作者：
R. Suardíaz;P. G. Jambrina;Laura Masgrau;Àngels González;E. Rosta;J. M. Lluch
通讯作者：
J. M. Lluch