EPR characterization of the mononuclear Cu‐containing Aspergillus japonicus quercetin 2,3‐dioxygenase reveals dramatic changes upon anaerobic binding of substrates

Abstract

Quercetin 2,3‐dioxygenase (2,3QD) is a copper‐containing dioxygenase that catalyses the oxidation of the flavonol quercetin to 2‐protocatechuoylphloroglucinol carboxylic acid with concomitant production of carbon monoxide. In contrast to iron dioxygenases, very little is known about copper dioxygenases. We have characterized 2,3QD from the fungus Aspergillus japonicus by electron paramagnetic resonance spectroscopy (EPR). At pH 6.0, 2,3QD shows a mixture of two EPR species. The major form has parameters typical of type 2 Cu sites (g// = 2.330, A// = 13.7 mT), the minor one has a more distorted geometry (g// = 2.290, A// = 12.5 mT). Anaerobic addition of the substrate quercetin results in a different, single species EPR spectrum with g// = 2.336, A// = 11.4 mT, parameters, which are in‐between those of the type 2 and type 1 Cu sites in the Peisach–Blumberg (g// vs. A//) plot. After turnover, a new EPR signal is observed, which is ascribed to the carboxylic acid ester product complex. This spectrum is similar to that of the native enzyme at pH 10.0 and has g‐tensor parameters suggesting a trigonal bipyramidal site. Of a variety of flavonoids studied, only flavonols are able to bind to the copper centre of 2,3QD. Nine flavonols with different hydroxylation patterns at the A‐ and B‐ring have been analysed. They cluster in two different regions of the Peisach–Blumberg plot and show that the presence of a 5‐OH group has a large effect on the A// parameter. Several differences are noted between A. japonicus 2,3QD and the enzyme from A. niger German Collection of Microorganisms 821.

Anaerobic enzyme⋅substrate structures provide insight into the reaction mechanism of the copper-dependent quercetin 2,3-dioxygenase

“…Endoperoxide intermediate 4 represents the most convenient structure to rationalize first the release of carbon monoxide and then the formation of the depside-bound form 5 of the enzyme, for which a putative signal corresponding to a trigonal bipyramidal geometry has been observed by EPR spectroscopy (10). Carbon monoxide liberation would occur with the breakage of one OOO and two COC bonds.…”

“…Quercetin 2,3-dioxygenase (2,3QD) belongs to the cupin superfamily (6,7) and is the only dioxygenase unambiguously known to rely on a mononuclear copper center for activity (8)(9)(10). It is a type 2 copper-dependent enzyme expressed by Aspergillus species when grown on complex aromatic compounds, such as rutin or quercetin.…”

“…The environment of the copper ion of Aspergillus japonicus 2,3QD has been probed by x-ray crystallography (6) and electron paramagnetic resonance (EPR) spectroscopy (10) and found to be heterogeneous. The crystal structure at 1.6 Å resolution shows the metal ion chiefly bound in a distorted tetrahedral geometry by three histidine residues (His 66 , His 68 , and His 112 ) and a water molecule.…”

Excess Production ofBacillus subtilisQuercetin 2,3-Dioxygenase Affects Cell Viability in the Presence of Quercetin

“…This assumption is also reinforced by the fact that QdoI is an intracellular enzyme, 24) and thus different from the secreted type of Aspergillus enzymes. 7,8,10) Correlation between QdoI activity and quercetin susceptibility…”

“…The gene encoding quercetin 2,3-dioxygenase has been cloned from A. japonicus, Streptomyces sp., and B. subtilis. 5,9,10) The crystal structures of the enzymes from A. japonicus and B. subtilis revealed that both enzymes belong to the bicupin family, which accommodates metal ions in their catalytic active sites, formed by characteristic -barrel domains. 8,11) While the fungal enzyme contains Cu 2þ , 8,12) the enzyme from B. subtilis is assumed to prefer Mn 2þ as a cofactor, 13) but its recombinant protein has been purified as an iron enzyme from Escherichia coli.…”