Deciphering the chemoselectivity of nickel-dependent quercetin 2,4-dioxygenase

The reaction mechanism and chemoselectivity of nickel-dependent quercetin 2,4-dioxygenase (2,4-QueD) have been investigated using the QM/MM approach. The protonation state of the Glu74 residue, a first-shell ligand of Ni, has been considered to be either neutral or deprotonated. QM/MM calculations predict that Glu74 must be deprotonated to rationalize the chemoselectivity and steer the 2,4-dioxygenolytic cleavage of quercetin, which harvests the experimentally-observed product, 2-protocatechuoylphloroglucinol carboxylic acid, coupled with the release of carbon monoxide. If the enzyme has a neutral Glu74 residue, the undesired 2,3-dioxygenolytic cleavage of quercetin becomes the dominant pathway, leading to the formation of α-keto acid. The calculations suggest that the reaction takes place via three major steps: (1) attack of the superoxide on the C2 of the substrate pyrone ring to generate a NiII-peroxide intermediate; (2) formation of the second C-O bond between C4 and the peroxide to produce a peroxide bridge; (3) simultaneous cleavage of the C2-C3, C3-C4, and O1-O2 bonds with the formation of 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide. The third step was found to be rate-limiting, with a barrier of 17.4 kcal mol-1, which is in very good agreement with the experimental kinetic data. For the second C-O bond formation, an alternative pathway is that the peroxide attacks the C3 of the substrate pyrone ring, leading to the formation of a four-membered ring intermediate, which then undergoes concerted C2-C3 and O1-O2 bond cleavages to produce an α-keto acid. This pathway is associated with a barrier of 30.6 kcal mol-1, which is much higher than the major pathway. When Glu74 is protonated, the 2,3-dioxygenolytic pathway, however, has a lower barrier (21.8 kcal mol-1) than the 2,4-dioxygenolytic pathway.