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

摘要

Quercetin 2,3-dioxygenase (2,3QD) is the only firmly established copper dioxygenase known so far. Depending solely on a mono-nuclear 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 com-plexed with the substrate kaempferol and the natural substrate quercetin have been determined at 1.90- and 1.75-A resolution, respectively. Flavonols coordinate to the cqpper ion as monoden-tate 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 Oε1 atom. The short distance of about 2.55 A between its Oε2 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.