Factors That Affect Oxygen Activation and Coupling of the Two Redox Cycles in the Aromatization Reaction Catalyzed by NikD, an Unusual Amino Acid Oxidase
Factors That Affect Oxygen Activation and Coupling of the Two Redox Cycles in the Aromatization Reaction Catalyzed by NikD, an Unusual Amino Acid Oxidase
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DOI：
10.1021/bi901056a
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发表时间：
2009-10-13
期刊：
影响因子：
2.9
通讯作者：
Jorns, Marilyn Schuman
中科院分区：
文献类型：
--
作者：
Kommoju, Phaneeswara-Rao;Bruckner, Robert C.;Jorns, Marilyn Schuman
NikD是一种黄素蛋白氧化酶，催化吡哌啶-2-羧酸酯(P2C)氧化生成吡哌甲酸酯，芳构化反应包括两个氧化还原循环和至少一个异构化步骤。Tyr258形成了一个“芳香笼子”的一部分，该笼子围绕着吡喃甲酸酯及其前体的环。Tyr258突变为Phe不会扰乱nikD的结构，但会影响两个氧化还原循环的耦合，导致周转率下降10倍。Tyr258Phe催化了P2C的定量两电子氧化，但只有60%的二氢吡啶甲酸酯中间体经历了第二个氧化还原循环来生成吡啶甲酸酯。这种突变不会影响另一种底物(3，4-脱氢-L-脯氨酸)的产品收率，该底物在单一的两电子氧化步骤中芳香化。野生型和突变型酶在将P2C氧化为二氢吡啶甲酸酯和还原酶异构化方面表现出相同的速率常数。二氢吡啶甲酸络合物。观察到的速度分别比突变人的周转速度快200倍和10倍。吡啶甲酸盐泡沫剂Tyr258Phe的释放速度是成交量的100倍。结合底物或产物的存在是野生型nikD氧活化的关键因素，通过观察到还原酶与吡啶甲酸酯、苯甲酸酯或1-环己烯酸酯(1-deaza-P2C类似物)的络合物的速度快10-75倍来判断这一点。还原的Tyr258Phe中心点1-环己烯酸络合物与氧的反应比野生型络合物少25倍。我们推测，Tyr258的突变导致活性部位动力学的细微变化，促进活性二氢吡啶甲酸中间体的释放，并扰乱与野生型nikD观察到的氧激活的有效同步。
NikD is a flavoprotein oxidase that catalyzes the oxidation of piperideine-2-carboxylate (P2C) to picolinate in a remarkable aromatization reaction comprising two redox cycles and at least one isomerization step. Tyr258 forms part of an "aromatic cage" that surrounds the ring in picolinate and its precursors. Mutation of Tyr258 to Phe does not perturb the structure of nikD but does affect the coupling of the two redox cycles and causes a 10-fold decrease in turnover rate. Tyr258Phe catalyzes a quantitative two-electron oxidation of P2C, but only 60% of the resulting dihydropicolinate intermediate undergoes a second redox cycle to produce picolinate. The mutation does not affect product yield with an alternate Substrate (3,4-dehydro-L-proline) that is aromatized in a single two-electron oxidation step. Wild-type and mutant enzymes exhibit identical rate constants for oxidation of P2C to dihydropicolinate and isomerization of a reduced enzyme . dihydropicolinate complex. The observed rates are 200- and 10-fold faster, respectively, than the mutant turnover rate. Release of picolinate froth Tyr258Phe is 100-fold faster than turnover. The presence of a bound substrate or product is a key factor in oxygen activation by wild-type nikD, as judged by the 10-75-fold faster rates observed for complexes of the reduced enzyme with picolinate, benzoate, or 1-cyclohexenoate, a 1-deaza-P2C analogue. The reduced Tyr258Phe center dot 1-cyclohexenoate complex is 25-fold less reactive with oxygen than the wild-type complex. We postulate that mutation of Tyr258 causes subtle changes in active site dynamics that promote release of the reactive dihydropicolinate intermediate and disrupt the efficient synchronization of oxygen activation observed with wild-type nikD.