Identification of the covalent flavin adenine dinucleotide-binding region in pyranose 2-oxidase from Trametes multicolor.
We present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. Pyranose 2-oxidase from the basidiomycete fungus Trametes multicolor, catalyzing C-2/C-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. No release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. The flavopeptide fragment resulting from tryptic/chymotryptic digestion of the purified enzyme was isolated by anion-exchange and reversed-phase high-performance liquid chromatography. The flavin type, attachment site, and mode of its linkage were determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy of the intact flavopeptide, without its prior enzymatic degradation to the central aminoacyl moiety. Mass spectrometry identified the attached flavin as flavin adenine dinucleotide (FAD). Post-source decay analysis revealed that the flavin is covalently bound to histidine residue in the peptide STHW, consistent with the results of N-terminal amino acid sequencing by Edman degradation. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy, resulting in the structure 8alpha-(N(3)-histidyl)-FAD.
Halada
Petr
P
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-142 20 Prague 4, Czech Republic. halada@biomed.cas.cz
Leitner
Christian
C
Sedmera
Petr
P
Haltrich
Dietmar
D
Volc
Jindrich
J
D001487:Basidiomycota / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D001665:Binding Sites; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D008968:Molecular Conformation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D020539:Sequence Analysis, Protein / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase