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 dioxyg enase proposed to belong to the alpha/beta hydrolase fold superfamily of en zymes. alpha/beta Hydrolases contain a highly conserved catalytic triad (nu cleophile-acidic residue-histidine). We previously identified a correspondi ng catalytically essential histidine residue in Qdo. However, as shown by a mino acid replacements through site-directed mutagenesis, nucleophilic and acidic residues of Qdo considered as possible triad residues were not absol utely required for activity. This suggests that Qdo does not contain the ca nonical catalytic triad of the alpha/beta hydrolase fold enzymes. Some radi cal trapping agents affected the Qdo-catalyzed reaction. A hypothetical mec hanism of Qdo-catalyzed dioxygenation of 1H-3-hydroxy-3-oxoquinoline is com pared with the dioxygenation of FMNH2 catalyzed by bacterial luciferase, wh ich also uses a histidine residue as catalytic base. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.