In several aerobic metabolic pathways, O2
is incorporated into organic compounds through monooxygenase- or dioxygenase-catalyzed reactions (Hayaishi, 1974
). For instance, oxygenation is often used to make lipids and particularly aromatic molecules amenable to further biochemical transformations. 1H
-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD; EC 18.104.22.168) from Arthrobacter nitroguajacolicus
Rü61a is a 32 kDa enzyme that is involved in the anthranilate pathway of quinaldine degradation (Bauer et al.
). It catalyses the O2
-dependent N-heteroaromatic ring cleavage of 1H
-3-hydroxy-4-oxoquinaldine to N
-acetylanthranilate and carbon monoxide (Fig. 1).
2,4-Dioxygenolytic ring cleavage of 1H-3-hydroxy-4-oxoquinaldine catalysed by HOD. Oxygen atoms from dioxygen are shown in larger bold font. This figure was created with MarvinSketch (ChemAxon).
Sequence analysis and secondary-structure prediction indicate that HOD belongs to the large superfamily of α/β-hydrolase-fold enzymes (Fischer et al.
; Nardini & Dijkstra, 1999
). HOD displays no sequence similarities to other known oxygenases, except for the 1H
-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO; EC 22.214.171.124) involved in 1H
-4-oxoquinoline degradation by Pseudomonas putida
33/1 (Qi et al.
). QDO catalyses a reaction very similar to that catalysed by HOD; namely, the dioxygenolytic N-heteroaromatic ring cleavage of 1H
-3-hydroxy-4-oxoquinoline to N
-formylanthranilate and carbon monoxide (Bauer et al.
). HOD and QDO share 37% identity at the sequence level and these two enzymes are the only dioxygenases that have been proposed to be members of the α/β-hydrolase-fold enzymes.
Oxygenases usually depend on a transition metal and/or organic cofactors for activity. This is a consequence of the fact that a direct reaction between the triplet ground-state O2
and singlet ground-state substrates to produce singlet-state products implies a violation of the conservation of the total angular momentum (Hamilton, 1974
). It is therefore a low-probability event. The presence of cofactors overcomes the spin-forbiddenness of the process. The mechanism used by nonhaem iron-dependent (Que, 1999
), copper-dependent (Steiner et al.
), flavin-dependent and pterin-dependent (Massey, 1994
; Palfey et al.
) oxygenases have been studied and various reviews are available that discuss the general mechanisms of oxygen and substrate activation in enzymatic oxygenation reactions. Biochemical and spectroscopic studies have shown that neither HOD nor QDO contain organic cofactors or stoichiometric amounts of any metal (Bauer et al.
; Fetzner, 2002
). The mechanism(s) employed by these dioxygenases are therefore very interesting from the viewpoint of fundamental enzymology.
The availability of three-dimensional information should increase our understanding of the catalytic mechanism employed by these intriguing cofactor-devoid dioxygenases as well as provide direct evidence about their fold. In this account, we report the crystallization and preliminary X-ray analysis of HOD.