Site-directed mutagenesis of potential catalytic residues in 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase, and hypothesis on the catalytic mechanism of 2,4-dioxygenolytic ring cleavage.

1H-3-Hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) is a cofactor-free dioxygenase proposed to belong to the alpha/beta hydrolase fold superfamily of enzymes. Alpha/beta Hydrolases contain a highly conserved catalytic triad (nucleophile-acidic residue-histidine). We previously identified a corresponding catalytically essential histidine residue in Qdo. However, as shown by amino acid replacements through site-directed mutagenesis, nucleophilic and acidic residues of Qdo considered as possible triad residues were not absolutely required for activity. This suggests that Qdo does not contain the canonical catalytic triad of the alpha/beta hydrolase fold enzymes. Some radical trapping agents affected the Qdo-catalyzed reaction. A hypothetical mechanism of Qdo-catalyzed dioxygenation of 1H-3-hydroxy-4-oxoquinoline is compared with the dioxygenation of FMNH2 catalyzed by bacterial luciferase, which also uses a histidine residue as catalytic base.