title: 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.
abstract: The cofactor of several flavoenzymes is autocatalytically bound to the polypeptide via a histidyl(N3)-(8alpha)-FAD linkage which makes the generation of apoenzyme difficult. We introduced an alternative covalent protein-FAD bond at the active site of 6-hydroxy-D-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.
authors.affiliation: Institut für Biochemie und Molekularbiologie, Frieburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Stoltz
authors.forename: H P
authors.initials: HP
authors.lastname: Henninger
authors.forename: R
authors.initials: R
authors.lastname: Brandsch
mesh_terms: D003067:Coenzymes / Q000737:chemistry / Q000235:genetics; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D015195:Drug Design; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000737:chemistry*; D005415:Flavins / Q000737:chemistry; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D013329:Structure-Activity Relationship
chemical_list: D003067:Coenzymes; D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; C090003:8-(methylsulfonyl)FAD; D006639:Histidine; C017123:8-chloro-FAD; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase; D003545:Cysteine