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

• Published in: FEBS letters Vol. 386; no. 2; pp. 194 - 196
• Main Authors: Stoltz, Michaela, Henninger, Hans-Peter, Brandsch, Roderich
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 20.05.1996
• Subjects: 6-hydroxy-d-nicotine oxidase; 6HDNO; Coenzymes - chemistry; Coenzymes - genetics; Covalent flavinylation; Cysteine - chemistry; Cysteine - genetics; Drug Design; Escherichia coli; FAD; Flavin-Adenine Dinucleotide - analogs & derivatives; Flavin-Adenine Dinucleotide - chemistry; Flavins - chemistry; Flavoenzyme; Histidine - chemistry; Histidine - genetics; Kinetics; Oxidoreductases - chemistry; Oxidoreductases - genetics; PAGE; Point Mutation; polyacrylamide gel electrophoresis; Site-directed mutagenesis; Structure-Activity Relationship; FAD; 6HDNO; Covalent flavinylation; PAGE; Flavoenzyme; Site-directed mutagenesis
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(96)00438-3

The cofactor of several flavoenzymes is autocatalytically bound to the polypeptide via a histidyl(N 3)-(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.