Flavonol 2,4-dioxygenase from Aspergillus niger DSM 821, a type 2 Cu-II-containing glycoprotein

Flavonol 2,4-dioxygenase, which catalyzes the cleavage of quercetin to carb on monoxide and 2-protocatechuoylphloroglucinol carboxylic acid, was purifi ed from culture filtrate of Aspergillus niger DSM 821 grown on rutin. It is a glycoprotein (46-54% carbohydrate) with N-linked oligo-mannose type glyc an 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 pur ified flavonol 2,4-dioxygenase was heated, it dissociated into three peptid es with apparent molecular masses of 63-67 kDa (L), 53-57 kDa (M), and 31-3 5 kDa (S), which occurred in a molar ratio of 1 : 1 : 1, suggesting a LMS s tructure. Crosslinking led to a 90-97 kDa species, concomitant with the dec rease of staining intensity of the 63-67 kDa (L) and the 31-35 kDa (S) pept ides. Analysis by matrix-assisted laser desorption/ionization-time of fligh t-MS showed peaks at m/z approximate to 69 600, m/z approximate to 51 700, and m/z approximate to 26 500 which are presumed to represent the three pep tides of flavonol 2,4-dioxygenase, and a broad peak at mit approximate to 9 6 300, which might correspond to the LS heterodimer as formed in the crossl inking 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 Cu-II to Cu-I ethylxanthate, is a potent inhibitor of flavonol 2,4- dioxygenase. Metal chelating agents (such as diethyldithiocarbamate, diphen ylthiocarbazone) strongly inhibited the enzymatic activity, but inactivatio n was not accompanied by loss of copper. The EPR spectrum of flavonol 2,4-d ioxygenase (as isolated) showed the characteristic parameters of a nonblue type 2 Cu-II protein. The Cu2+ is assumed to interact with four nitrogen li gands, and the Cu-II complex has a (distorted) square planar geometry.