Monooxygenases and dioxygenases catalyse the incorporation of oxygen into organic compounds in several aerobic metabolic pathways. 1H
-4-Oxoquinoline is degraded by Pseudomonas putida
the anthranilate pathway. An intermediate step in this pathway is the ring oxygenolysis of 1H
-3-hydroxy-4-oxoquinoline to form carbon monoxide and N
-formylanthranilate. This step is catalysed by 1H
-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO; EC 220.127.116.11). Arthrobacter nitroguajacolicus
Rü61a (formerly A. ilicis
Rü61a) possesses an enzyme, 1H
-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD; EC 18.104.22.168; Fischer et al.
), that catalyzes an analogous reaction (Fig. 1).
The reactions catalysed by QDO (R = H) and HOD (R = CH3). The substrates are 1H-3-hydroxy-4-oxoquinoline (R = H) and 1H-3-hydroxy-4-oxoquinaldine (R = CH3).
Most oxygenases contain metal ions (usually copper or iron) or flavins to activate oxygen. QDO and HOD have no requirement for such cofactors (Fetzner, 2002
). Kinetic analysis of HOD indicates that the enzyme operates by a compulsory-order ternary-complex mechanism, with binding of the heterocyclic substrate preceding that of oxygen (Frerichs-Deeken et al.
QDO is a 264-amino-acid protein of molecular weight 30 kDa. Analysis of the amino-acid sequence of QDO indicates that it is a member of the α/β-hydrolase superfamily of enzymes (Fischer et al.
). The canonical fold has a central β-sheet with strand β2 antiparallel to the rest. The connection of strand order is +1, +2, −1x, +2x, +1x, +1x (Heikinheimo et al.
). These enzymes contain a catalytic triad consisting of a nucleophile, a histidine and an acidic residue. The nucleophile can be a serine, cysteine or aspartate residue. In QDO, the catalytic histidine has been identified (Fischer & Fetzner, 2000
). Site-directed mutagenesis of QDO indicates that this residue is absolutely required for catalytic activity (Fischer & Fetzner, 2000
In order to understand the catalytic mechanism of QDO, we have undertaken to solve the structure of this enzyme. Here, we report the expression, purification, crystallization and preliminary diffraction data for QDO. The crystallization of HOD is described in the accompanying paper (Steiner et al.