title: Site-directed mutagenesis of the FAD-binding histidine of 6-hydroxy-D-nicotine oxidase. Consequences on flavinylation and enzyme activity.
abstract: In 6-hydroxy-D-nicotine oxidase (6-HDNO) FAD is covalently bound to His71 of the polypeptide chain by an 8 alpha-(N3-histidyl)-riboflavin linkage. The FAD-binding histidine was exchanged by site-directed mutagenesis to either a Cys- or Tyr-residue, two amino acids known to be involved in covalent binding of FAD in other enzymes, or to a Ser-residue. None of the amino acid replacements for His71 allowed covalent FAD incorporation into the 6-HDNO polypeptide. Thus, the amino acid residues involved in covalent FAD-binding require a specific polypeptide surrounding in order for this modification to proceed and cannot be replaced with each other. Enzyme activity was completely abolished with Tyr in place of His71. 6-HDNO activity with non-covalently bound FAD was found with 6-HDNO-Cys and to a lesser extent also with 6-HDNO-Ser. However, the Km values for 6-HDNO-Cys and 6-HDNO-Ser were increased approximately 20-fold as compared to 6-HDNO-His. Both mutant enzymes, in contrast to the wild-type enzyme, needed additional FAD in the enzymatic assay (50 microM for 6-HDNO-Ser and 10 microM for 6-HDNO-Cys) for maximal enzyme activity.
authors.affiliation: Biochemisches Institut, Universität Freiburg, FRG.
authors.forename: L
authors.initials: L
authors.lastname: Mauch
authors.forename: V
authors.initials: V
authors.lastname: Bichler
authors.forename: R
authors.initials: R
authors.lastname: Brandsch
mesh_terms: D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids
chemical_list: D003062:Codon; D005182:Flavin-Adenine Dinucleotide; D006639:Histidine; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase