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首页> 外文期刊>Acta crystallographica.Section D. Biological crystallography >A hydrogen-bonding network is important for oxidation and isomerization in the reaction catalyzed by cholesterol oxidase
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A hydrogen-bonding network is important for oxidation and isomerization in the reaction catalyzed by cholesterol oxidase

机译：氢键网络是很重要的氧化和异构化反应由胆固醇氧化酶催化

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摘要

Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of 3betahydroxysteroids. Structural and mutagenesis studies have shown that Asn485 plays a key role in substrate oxidation. The side chain makes an NH- -pi interaction with the reduced form of the flavin cofactor. A N485D mutant was constructed to further test the role of the amide group in catalysis. The mutation resulted in a 1800-fold drop in the overall k_(cat). Atomic resolution structures were determined for both the N485L and N485D mutants. The structure of the N485D mutant enzyme (at 1.0 A resolution) reveals significant perturbations in the active site. As predicted, Asp485 is oriented away from the flavin moiety, such that any stabilizing interaction with the reduced flavin is abolished. Metl22 and Glu361 form unusual hydrogen bonds to the functional group of Asp485 and are displaced from the positions they occupy in the wild-type active site. The overall effect is to disrupt the stabilization of the reduced FAD cofactor during catalysis. Furthermore, a narrow transient channel that is shown to form when the wild-type Asn485 forms the NH- -pi interaction with FAD and that has been proposed to function as an access route of molecular oxygen, is not observed in either of the mutant structures, suggesting that the dynamics of the active site are altered.
机译：胆固醇氧化酶是黄素酶催化的氧化和异构化3 betahydroxysteroids。研究表明,Asn485起着关键的作用在底物氧化。NH - -π交互的简化形式黄素代数余子式。为了进一步测试中的酰胺基的作用催化。总体下降k_(猫)。结构为N485L和决定N485D突变体。1.0酶(分辨率)揭示了意义重大扰动的活性部位。Asp485离黄素的一部分,这样,任何稳定交互减少黄素是废除。不寻常的氢键形式功能群Asp485和背井离乡的他们在野生型活动占据位置网站。稳定减少时尚代数余子式的催化。当野生型通道形成显示Asn485形成NH - -π交互与时尚提出了函数作为一个访问路线的分子氧,没有观察到这两个突变体结构,表明活性部位的动态改变。

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