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

FEBS Letters

Henninger, Hans-Peter¹  Stoltz, Michaela¹  Brandsch, Roderich¹ 

[1] Institut für Biochemie und Molekularbiologie, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany

关键词: Flavoenzyme;    Site-directed mutagenesis;    Covalent flavinylation;    FAD;    6HDNO;    6-hydroxy-d-nicotine oxidase;    PAGE;    polyacrylamide gel electrophoresis;   

DOI  :  10.1016/0014-5793(96)00438-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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-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.

【 授权许可】

Unknown