Flavonol 2,4-dioxygenase from Aspergillus niger DSM 821, a type 2 CuII-containing glycoprotein.
Flavonol 2,4-dioxygenase, which catalyzes the cleavage of quercetin to carbon monoxide and 2-protocatechuoyl-phloroglucinol carboxylic acid, was purified from culture filtrate of Aspergillus niger DSM 821 grown on rutin. It is a glycoprotein (46-54% carbohydrate) with N-linked oligo-mannose type glycan chains. The enzyme was resolved in SDS polyacrylamide gels in a diffuse protein band that corresponded to a molecular mass of 130-170 kDa. When purified flavonol 2,4-dioxygenase was heated, it dissociated into three peptides with apparent molecular masses of 63-67 kDa (L), 53-57 kDa (M), and 31-35 kDa (S), which occurred in a molar ratio of 1:1:1, suggesting a LMS structure. Crosslinking led to a 90-97 kDa species, concomitant with the decrease of staining intensity of the 63-67 kDa (L) and the 31-35 kDa (S) peptides. Analysis by matrix-assisted laser desorption/ionization-time of flight-MS showed peaks at m/z approximately 69 600, m/z approximately 51 700, and m/z approximately 26 500 which are presumed to represent the three peptides of flavonol 2,4-dioxygenase, and a broad peak at m/z approximately 96 300, which might correspond to the LS heterodimer as formed in the crosslinking reaction. Based on the estimated molecular mass of 148 kDa, 1 mol of enzyme contained 1.0-1.6 mol of copper. Ethylxanthate, which specifically reduces CuII to CuI ethylxanthate, is a potent inhibitor of flavonol 2,4-dioxygenase. Metal chelating agents (such as diethyldithiocarbamate, diphenylthiocarbazone) strongly inhibited the enzymatic activity, but inactivation was not accompanied by loss of copper. The EPR spectrum of flavonol 2,4-dioxygenase (as isolated) showed the characteristic parameters of a nonblue type 2 CuII protein. The Cu2+ is assumed to interact with four nitrogen ligands, and the CuII complex has a (distorted) square planar geometry.
Hund
H K
HK
Institut für Mikrobiologie, Universität Hohenheim, Stuttgart, Germany.
Breuer
J
J
Lingens
F
F
Hüttermann
J
J
Kappl
R
R
Fetzner
S
S
D001234:Aspergillus niger / Q000201:enzymology* / Q000254:growth & development; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000032:analysis*; D003432:Cross-Linking Reagents; D049308:Dioxygenases; D004578:Electron Spin Resonance Spectroscopy; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010105:Oxygenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012431:Rutin / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014462:Ultrafiltration
D003432:Cross-Linking Reagents; D006023:Glycoproteins; D046911:Macromolecular Substances; D012431:Rutin; D003300:Copper; D010105:Oxygenases; D049308:Dioxygenases; C121201:flavonol 2,4-dioxygenase