Identification of the covalently bound flavin prosthetic group of cholesterol oxidase.
Highly purified preparations of cholesterol oxidase from Schizophyllum commune contain a covalently bound flavin component. A flavin peptide has been obtained by digestion with trypsin-chymotrypsin and purification on a column of phosphocellulose. Digestion with nucleotide pyrophosphatase results in increased fluorescence at pH 3.4 and release of 5'-adenylate, showing that the flavin is in the dinucleotide form. The absorption spectrum of the flavin peptide shows the hypsochromic shift of the second absorption band characteristic of 8 alpha-substituted flavins. The fluorescence at pH 7 is extensively quenched even in the mononucleotide form, with a pKa at pH 5.8 in the flavin peptide and at 5.05 following acid hydrolysis to the aminoacyl flavin level. This suggests that histidine is the amino acid substituted at the 8 alpha position of the flavin and that N(1) of the imidazole ring is the site of attachment. These data, the reduction of the flavin by borohydride, and comparison of the mobilities in high voltage electrophoresis at two pH values with N(1)- and N(3)-histidyl riboflavin and their 2',5'-anhydro forms shows that the prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD.
D015096:3-Hydroxysteroid Dehydrogenases; D000363:Agaricales / Q000201:enzymology*; D002789:Cholesterol Oxidase; D005486:Flavin Mononucleotide; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000032:analysis; D012567:Schizophyllum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; D006639:Histidine; D005486:Flavin Mononucleotide; D015096:3-Hydroxysteroid Dehydrogenases; D002789:Cholesterol Oxidase