Structural basis for cofactor-independent dioxygenation of N-heteroaromatic compounds at the alpha/beta-hydrolase fold

Abstract

Enzymatic catalysis of oxygenation reactions in the absence of metal or organic cofactors is a considerable biochemical challenge. The CO-forming 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) from Arthrobacter nitroguajacolicus Rü61a and 1-H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) from Pseudomonas putida 33/1 are homologous cofactor-independent dioxygenases involved in the breakdown of N-heteroaromatic compounds. To date, they are the only dioxygenases suggested to belong to the alpha/beta-hydrolase fold superfamily. Members of this family typically catalyze hydrolytic processes rather than oxygenation reactions. We present here the crystal structures of both HOD and QDO in their native state as well as the structure of HOD in complex with its natural 1-H-3-hydroxy-4-oxoquinaldine substrate, its N-acetylanthranilate reaction product, and chloride as dioxygen mimic. HOD and QDO are structurally very similar. They possess a classical alpha/beta-hydrolase fold core domain additionally equipped with a cap domain. Organic substrates bind in a preorganized active site with an orientation ideally suited for selective deprotonation of their hydroxyl group by a His/Asp charge-relay system affording the generation of electron-donating species. The "oxyanion hole" of the alpha/beta-hydrolase fold, typically employed to stabilize the tetrahedral intermediate in ester hydrolysis reactions, is utilized here to host and control oxygen chemistry, which is proposed to involve a peroxide anion intermediate. Product release by proton back transfer from the catalytic histidine is driven by minimization of intramolecular charge repulsion. Structural and kinetic data suggest a nonnucleophilic general-base mechanism. Our analysis provides a framework to explain cofactor-independent dioxygenation within a protein architecture generally employed to catalyze hydrolytic reactions.

Fig. 1.

(A) Scheme of the reaction catalyzed by the bacterial CO-forming cofactor-devoid dioxygenases. HOD catalyzes the conversion of QND (R = CH₃) to NAA, whereas QDO catalyzes the conversion of 1-H-3-hydroxy-4-oxoquinoline (R = H, QNL) to N-formylanthranilate. In the dioxygenolytic reaction, the ring A of the N-heteroatomic substrate is disrupted with formation of carbon monoxide as by-product. (B) Cartoon representation of HOD with the α/β-hydrolase fold core domain and cap domain shown in light gray and dark gray, respectively. Residues of the catalytic triad are shown in ball-and-stick representation. Secondary structure elements are labeled. N and C indicate the N and C termini, respectively. (All figures except Figs. 1A and 4 were prepared with Pymol and Adobe Illustrator.)

Fig. 4.

Proposed reaction mechanism for the cofactor-independent dioxygenation of N-heteroaromatic substrates at the α/β-hydrolase fold.

Fig. 2.

(A) Sliced-surface top-view (from the cap domain) highlighting access to the active site cavity. The centroid of the channel leading to the catalytic center is shown as a yellow trace. Residues below the slicing planes are shown as ribbons with core domain and cap domain structural elements in light and dark gray, respectively. Residues of the catalytic triad are shown in ball-and-stick mode. The molecular surface is colored according to the electrostatic potential. Positive and negative potential are shown in blue and red, respectively. (B) Sliced-surface back-view (opposite to the channel entrance) showing a vertical section of the active site cavity corresponding to the middle of the basin. The dotted line indicates the approximate boundary between the core domain and the cap domain. Residues Trp160 and Ile192 of the cap domain, which play an important role in shaping the catalytic pocket, are shown in stick representation. (C– E) Magnified view of the active site section framed in (B) by the gray rectangle with the substrate (C), product (D), and chloride ion as O₂ mimic (E) bound to HOD. For clarity, bound molecules are shown unclipped. Tunnel and electrostatic potential calculations were performed with the programs MOLE (34) and APBS (35), respectively.

Fig. 3.

(A) Active site of the anaerobic HOD·QND (enzyme·substrate) complex. (B) Active site of the HOD·NAA (enzyme·complex) complex. (C) Active site of the HOD·Cl^- (enzyme·O₂ mimic) complex with superimposed a QND substrate molecule. The simulated-annealing OMIT Fo-Fc electron density for the bound HQD, NAA, and Cl^- molecules contoured at the 3.0σ level is shown in green, orange, and cyan, respectively. In (C), an NCS-averaged anomalous difference map (shown in yellow) contoured at the 5.5σ level highlights the location of the chloride ion and nearby sulfur atoms. An approximately 12-ų cavity available below the QND molecule is shown in blue as a transparent surface. Side chains of residues lining the active site cavity are shown in stick representation, with residues within 4.0 Å of the substrate and product molecules labeled in green and orange, respectively. Residues of the triad are represented in ball-and-stick representation. Hydrogen bonds are shown as gray dashed lines. The red sphere represents a water molecule. Distances are in angstroms.