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

Abstract

Quercetin 2,3-dioxygenase (2,3QD) is the only firmly established copper dioxygenase known so far. Depending solely on a mononuclear Cu center, it catalyzes the breakage of the O-heterocycle of flavonols, producing more easily degradable phenolic carboxylic acid ester derivatives. In the enzymatic process, two C—C bonds are broken and concomitantly carbon monoxide is released. The x-ray structures of Aspergillus japonicus 2,3QD anaerobically complexed with the substrate kaempferol and the natural substrate quercetin have been determined at 1.90- and 1.75-Å resolution, respectively. Flavonols coordinate to the copper ion as monodentate ligands through their 3OH group. They occupy a shallow and overall hydrophobic cavity proximal to the metal center. As a result of a van der Waals contact between the most outward flavonol A-ring and Pro 164 , a flexible loop in front of the active site becomes partly ordered. Interestingly, flavonols bound to 2,3QD are bent at the C2 atom, which is pyramidalized. The increased local sp 3 character at this atom may stabilize a carbon-centered radical activated for dioxygen attack. Glu 73 coordinates the copper through its Oɛ1 atom. The short distance of about 2.55 Å between its Oɛ2 atom and the flavonol O3 atom suggests that a hydrogen bond exists between the two atoms, indicating that Glu 73 can act as a base in flavonol deprotonation and that it retains the proton. Structure-based geometric considerations indicate O 2 binding to the flavonol C2 atom as the preferred route for flavonol dioxygenation.