The crystal structures of the copper-dependent Aspergillus japonicusquercetin 2,3-dioxygenase (2,3QD) complexed with the inhibitors diethyldithiocarbamate (DDC) and kojic acid (KOJ) are reported at 1.70 and 2.15 Å resolution, respectively. Both inhibitors asymmetrically chelate the metal center and assume a common orientation in the active site cleft. Their molecular plane blocks access to the inner portion of the cavity which is lined by the side chains of residues Met51, Thr53, Phe75, Phe114, and Met123 and which is believed to bind the flavonol B-ring of the natural substrate. The binding of the inhibitors brings order into the mixed coordination observed in the native enzyme. DDC and KOJ induce a single conformation of the Glu73 side chain, although in different ways. In the presence of DDC, Glu73 is detached from the copper ion with its carboxylate moiety pointing away from the active site cavity. In contrast, when KOJ is bound, Glu73 ligates the Cu ion through its O 1 atom with a monodentate geometry. Compared to the native coordinating conformation, this conformation is approximately 90 ° rotated about the ø3 angle. This latter Glu73 conformation is compatible with the presence of a bound substrate. Dioxygenases are enzymes that catalyze the incorporation of both atoms of molecular oxygen into organic substrates. They take part in the metabolism of biomolecules as different as amino acids, lipids, nucleic acids, and even carbohydrates (1). Their biological importance relates in particular, however, to their ability to catalyze the degradation of aromatic compounds ( 1-3). Dioxygenases are often mononuclear non-heme ironcontaining enzymes ( 4). In the thoroughly investigated extradioland intradiol-type catechol dioxygenases, structural and spectroscopic studies have elucidated how the iron center is exploited to accomplish catalysis (for reviews, see refs 2 and 5). Extradiol-type dioxygenases utilize an Fe 2+ ion to ligate and to directly activate O 2, whereas the intradiol-type enzymes employ Fe 3+ to activate their substrate prior to dioxygen attack. Although their employed strategies are different, both enzyme types utilize the metal cofactor to circumvent the spin barrier that prohibits the direct reaction of ground-state triplet dioxygen with singlet-state organic substrates. Dioxygenases containing other metal centers (Cu ( 6-9), Mn (10), Mg (11)) have also been reported. They have, however, not been studied as extensively as the ironcontaining enzymes. Quercetin 2,3-dioxygenase (2,3QD) 1 is the only dioxygenase unambiguously known to contain copper ( 7, 9). It cleaves theO-heterocycle of polyphenolic flavonols (Figure 1a) which represent a major class of flavonoids (12). These compounds are important dietary components and have attracted considerable attention in the past decade owing to their antioxidizing properties ( 13-15). Recently, our group has elucidated the X-ray structure of 2,3QD fromAspergillus japonicusat 1.6 Å resolution (PDB code 1JUH ( 16)) providing the first structural view of a noniron dioxygenase. The enzyme is a homodimer of ∼100 kDa. The dimerization is probably not of functional relevance because the copper centers are about 40 Å apart, and no residues from the other subunit are nearby. The monomer contains two domains, an N-terminal domain (residues 1-145) and a C-terminal domain (residues 206 -350), which are joined by a long stretch of 60 amino acids (residues 146 205). The two domains share a common jelly roll motif. A large shallow hydrophobic cavity located in the N-terminal domain hosts the metallocenter. On the basis of a sequence analysis and its three-dimensional fold, the enzyme has been found to belong to the cupin superfamily ( 16). The coordination environment of the copper ion in 2,3QD is complex (Figure 2a). The metal is chiefly bound in a distorted tetrahedral geometry by three histidine residues † The research described here was supported by The Netherlands Foundation for Chemical Research (CW) with the financial aid from The Netherlands Foundation for Scientific Research (NWO). The EU supported the work at the synchrotron sites through the HCMP Access to Large Installations Program. * Corresponding author. Phone: +31-50-3634381. Fax:+31-503634800. E-mail: bauke@chem.rug.nl. ‡ University of Groningen. § Unilever Research Vlaardingen. 1 Abbreviations: 2,3QD, quercetin 2,3-dioxygenase; DDC, sodium diethyldithiocarbamate; EPR, electron paramagnetic resonance; E ‚S, enzyme‚flavonol complex; EXAFS, extended X-ray absorption fine structure; KOJ, kojic acid (5-hydroxy-2-(hydroxymethyl)-4 H-pyran-4one); QUE, quercetin (5,7,3 ′,4′-tetrahydroxy flavonol); XAS, X-ray absorption spectroscopy. 7955 Biochemistry2002,41, 7955-7962 10.1021/bi0159736 CCC: $22.00 © 2002 American Chemical Society Published on Web 06/01/2002 (His66, His68, and His112) and a water molecule (Wat td). However, a minor trigonal bipyramidal/square pyramidal coordination is also observed, in which the water molecule (Wattb) is positioned somewhat further away from the Cu ion (Cu-Wattb ) 2.4 Å, Cu-Wattd ) 2.2 Å) and the side chain of Glu73 additionally coordinates the metal. The latter coordination is particularly interesting because carboxylate ligation has never before been observed in natural copper proteins ( 18-20). To date, no structural data are available on 2,3QD substrate or inhibitor complexes. In this report, we present the crystal structures of 2,3QD inhibited with diethydithiocarbamate (2,3QD‚DDC) along with that of its complex with kojic acid (2,3QD‚KOJ). The molecular structures of DDC and KOJ are presented in Figure 1b. Our crystallographic study shows the molecular basis for a common inhibitory mode and indicates that the binding of these effectors induces order in the copper coordination. Additionally, valuable mechanistic insights, particularly on the role of Glu73, are inferred from this work. MATERIALS AND METHODS 2,3QD Preparation and Purification. 2,3QD was overexpressed and purified as previously described ( 16). Protein purity was ascertained by SDS -PAGE gel electrophoresis. The metal site was found to be fully occupied by atomic absorption spectrometry using a Perkin-Elmer model Plasma 1000. Crystallization and Soaking Experiments. For crystallization purposes, the purified 2,3QD glycoenzyme (ca. 25% carbohydrate content, w/w) was treated with Endoglycosidase-H (Boehringer-Mannheim, Almere, The Netherlands) following the protocol indicated by the manufacturer. The released carbohydrates were removed by gel filtration chromatography on a Pharmacia Superdex G75 column. This procedure reduced the residual glycosylation to about 3 -5% (w/w). An activity assay, carried out according to the procedure of Oka et al. ( 21), indicated that the deglycosylation had no adverse effect on the catalytic efficiency of

The crystal structures of the copper-dependent Aspergillus japonicus quercetin 2,3-dioxygenase (2,3QD) complexed with the inhibitors diethyldithiocarbamate (DDC) and kojic acid (KOJ) are reported at 1.70 and 2.15 A resolution, respectively. Both inhibitors asymmetrically chelate the metal center and assume a common orientation in the active site cleft. Their molecular plane blocks access to the inner portion of the cavity which is lined by the side chains of residues Met51, Thr53, Phe75, Phe114, and Met123 and which is believed to bind the flavonol B-ring of the natural substrate. The binding of the inhibitors brings order into the mixed coordination observed in the native enzyme. DDC and KOJ induce a single conformation of the Glu73 side chain, although in different ways. In the presence of DDC, Glu73 is detached from the copper ion with its carboxylate moiety pointing away from the active site cavity. In contrast, when KOJ is bound, Glu73 ligates the Cu ion through its O(epsilon)(1) atom with a monodentate geometry. Compared to the native coordinating conformation, this conformation is approximately 90 degrees rotated about the chi(3) angle. This latter Glu73 conformation is compatible with the presence of a bound substrate.

Citation for published version (APA): Kooter, I. M., Steiner, R. A., Dijkstra, B. W., Noort, P. I. V., Egmond, M. R., & Huber, M. (2002). EPR characterization of the mononuclear Cu-containing Aspergillus japonicus quercetin 2,3-dioxygenase reveals dramatic changes upon anaerobic binding of substrates. European Journal of Biochemistry, 269(12), 2971-2979. https://doi.org/10.1046/j.1432-1033.2002.02973.x

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.