title: Lysine can replace arginine 67 in the mediation of covalent attachment of FAD to histidine 71 of 6-hydroxy-D-nicotine oxidase.
abstract: The requirements for FAD-attachment to His71 of 6-hydroxy-D-nicotine oxidase (6-HDNO) were investigated by site-directed mutagenesis. The following amino acid replacements were introduced into the sequence Arg67-Ser68-Gly69-Gly70-His71 of the 6-HDNO-polypeptide: 1) Arg67 was replaced with Ala (A1 mutant); 2) Ser68 was replaced with Ala (A2 mutant); and 3) Arg67 was replaced with Lys (K mutant). The substitution in mutant A2 had no effect on flavinylation, measured as [14C]FAD incorporation into apo-6-HDNO. Replacement of Arg67 with Ala prevented, but replacement with Lys permitted the flavinylation of His71. Mutant A1 showed no 6-HDNO activity, whereas the replacement of Ser with Ala in mutant A2 had only a slight effect on 6-HDNO activity. The substitution of Lys for Arg67, however, reduced the specific 6-HDNO activity in extracts of Escherichia coli cells expressing the mutant polypeptide from 50.3 to 17.5 milliunits/mg protein. It is concluded that a basic amino acid residue (Arg67 or Lys67) is required to mediate the attachment of FAD to His71, and while Lys can substitute for Arg67 in this function, it can only partially replace Arg67 in the enzyme reaction mechanism of 6-HDNO.
authors.affiliation: Biochemisches Institut, Universität Freiburg, Federal Republic of Germany.
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: D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006639:Histidine; D008239:Lysine; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids
chemical_list: D015345:Oligonucleotide Probes; D005182:Flavin-Adenine Dinucleotide; D006639:Histidine; D001120:Arginine; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase; D008239:Lysine