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首页> 外文期刊>FEBS Letters >The design of an alternative, covalently flavinylated 6‐hydroxy‐d‐nicotine oxidase by replacing the FAD‐binding histidine by cysteine and reconstitution of the holoenzyme with 8‐(methylsulfonyl)FAD
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The design of an alternative, covalently flavinylated 6‐hydroxy‐d‐nicotine oxidase by replacing the FAD‐binding histidine by cysteine and reconstitution of the holoenzyme with 8‐(methylsulfonyl)FAD

机译：通过半胱氨酸替换FAD结合的组氨酸和用8-（甲基磺酰基）FAD重建全酶的替代，共价转移的6-羟基-NICOTIN氧化酶的设计

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>The cofactor of several flavoenzymes is autocatalytically bound to the polypeptide via a histidyl(N3)-(8α)-FAD linkage which makes the generation of apoenzyme difficult. We introduced an alternative covalent protein-FAD bond at the active site of 6-hydroxy- class="smallCaps">n-nicotine oxidase (6HDNO) by replacing the FAD-binding histidine with cysteine. The resulting mutant enzyme was expressed with noncovalently attached cofactor. Incubation with 8-(methylsulfonyl)FAD, and less efficiently with 8-chloro-FAD, resulted in the spontaneous replacement of the noncovalently bound FAD by the flavin derivative and the formation of an 8-(N-acetylcysteinyl)FAD linkage. The flavinylated 6HDNO.cys exhibited close to wild-type activity levels. This strategy may be generally applicable to the attachment of artificially designed flavin derivatives to the active site of covalently flavinylated enzymes.
机译：>几种黄酮的辅因子通过组氨基（n 3 ） - （8α）-fad连杆自动致催化与多肽结合，这使得迄今为止难以产生。通过用半胱氨酸取代FAD结合的组氨酸，在6-羟基 - <跨度=“小型胶囊”> N - 训练酶（6HDNO）中介绍了替代的共价蛋白质 - FAD键。用非共价连接的辅助球表达所得突变酶。与8-（甲基磺酰基）的产品孵育，较少有效地用8-氯，导致Flavin衍生物的非共价结合的非共价结合，并形成8 - （ N - 乙酰胞嘧啶基）FAD键。黄丙烷化的6hdno.cys表现出近似野生型活性水平。该策略通常适用于将人工设计的黄素衍生物的附着到共价黄丙烷化酶的活性位点。

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