title: The conformational change induced by FAD in covalently flavinylated 6-hydroxy-D-nicotine oxidase does not require (8alpha)FAD-(N3)histidyl bond formation.
abstract: The contribution of (8alpha)-(N3)histidyl bond formation to the conformation of covalently flavinylated proteins was investigated by trypsin treatment of wild type and mutant versions of a model enzyme, 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter nicotinovorans. In the absence of FAD, apo-6-HDNO exhibited a conformation exposing a protease accessible site. Holoenzyme formation through FAD-attachment to His71 induced a conformational change in the protein that shielded the trypsin recognition site. This conformational change, however, did not require FAD-histidyl bond formation since trypsin resistance was also exhibited by a 6-HDNO.Cys71 mutant protein which was unable to bind FAD covalently. Replacement of Arg67, an amino acid residue supposed to be essential in flavinylation, by Ala rendered the protein protease sensitive as did replacement of Pro73 by Ala. These amino acids apparently play an essential role in stabilizing the native protein conformation. The inability to reach the native conformation also prevented FAD attachment, indicating that a specific conformation of the protein is a prerequisite for FAD-histidyl bond formation. Deletion of Phe448 and Arg449 from the 458 amino acid residues-containing enzyme resulted in complete protease sensitivity, demonstrating that flavinylation takes place posttranslationally.
authors.affiliation: Institut für Biochemie und Molekularbiologie, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Stoltz
authors.forename: R
authors.initials: R
authors.lastname: Brandsch
mesh_terms: D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012680:Sensitivity and Specificity; D014357:Trypsin
chemical_list: D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; C021413:6-hydroxy-D-nicotine oxidase; D014357:Trypsin