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 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 ≈ 69 600, m / z ≈ 51 700, and m / z ≈ 26 500 which are presumed to represent the three peptides of flavonol 2,4‐dioxygenase, and a broad peak at m / z ≈ 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 Cu II to Cu I  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 Cu II protein. The Cu 2+ is assumed to interact with four nitrogen ligands, and the Cu II complex has a (distorted) square planar geometry.