Crystal structure of the Anopheles gambiae 3-hydroxykynurenine transaminase

Abstract

In Anopheles gambiae, the vector for the most deadly malaria parasite Plasmodium falciparum, xanthurenic acid (XA) plays a key role in parasite gametogenesis and fertility. In mosquitoes, XA is produced by transamination of 3-hydroxykynurenine (3-HK), a reaction that represents the main route to prevent the accumulation of the potentially toxic 3-HK excess. Interfering with XA metabolism in A. gambiae therefore appears an attractive avenue for the development of malaria transmission-blocking drugs and insecticides. We have determined the crystal structure of A. gambiae 3-HK transaminase in its pyridoxal 5'-phosphate form and in complex with a newly synthesized competitive enzyme inhibitor. Structural inspection of the enzyme active site reveals the key molecular determinants for ligand recognition and catalysis. Major contributions toward inhibitor binding are provided by a salt bridge between the inhibitor carboxylate and Arg-356 and by a remarkable hydrogen bond network involving the anthranilic moiety of the inhibitor and backbone atoms of residues Gly-25 and Asn-44. This study may be useful for the structure-based design of specific enzyme inhibitors of potential interest as antimalarial agents.

Scheme 1.

Scheme of the two-step reaction catalyzed by the 3-HK transaminase. The first half of the reaction is irreversible.

Fig. 1.

Ag-HKT molecular architecture. (A) Stereo ribbon representation of the Ag-HKT subunit. The N-terminal extension, the large domain, and the small domain are colored in white, blue, and magenta, respectively. The loop protruding into the active site and carrying the cisPro-24-Gly-25 motif is labeled L1. The deep cleft hosting the enzyme active site is placed at the domain interface, where the PLP cofactor and the inhibitor molecules are shown in ball-and-stick representation. The N and the C termini are indicated, as are the helices connected by the loops involved in dimer stabilization. (B) Ribbon representation of the functional Ag-HKT homodimer seen along the dyad axis. The two subunits are colored white and green; PLP and inhibitor molecules are drawn in ball-and-stick representation. The N and the C termini are indicated. The figure was generated with the program molscript (50).

Fig. 2.

The Ag-HKT catalytic site. (A) Stereoview of the active site of the Ag-HKT:PLP structure. The PLP cofactor, the glycerol molecule, and protein residues involved in cofactor binding are depicted as ball and stick. (B) Stereo view of the active site in the Ag-HKT:INI structure. Only the 2F_o−F_c electron density map covering the 4-(2-aminophenyl)-4-oxobutyric acid inhibitor (INI) is shown contoured at 1.2σ level. The figure was generated with the program bobscript (51).

Fig. 3.

Closeup view of the enzyme active site in the Ag-HKT:INI structure. The protein portions building up the active site and contributed by the two subunits of the functional homodimer are shown in a ribbon representation and colored blue and magenta. The PLP cofactor, the inhibitor molecule, and the protein residues involved in ligand binding are shown. The inhibitor C2 atom equivalent to the Cα of the physiological amino acid substrate is indicated. The major interactions established between the inhibitor and residues of Ag-HKT are indicated as dotted lines. The figure was generated by using pymol (www.pymol.org).

Fig. 4.

Closeup view of the protein region surrounding the loop protruding into the active site and carrying the cisPro24-Gly-25 motif participating in ligand binding. The PLP cofactor, the inhibitor molecule, and the cisPro-24-Gly-25 motif are drawn as ball-and-stick representation. The coloring and the orientation are as in Fig. 1A. The figure was generated with the program molscript (50).

Scheme 2.

Schematic view of the Ag-HKT active site. The key interactions involved in inhibitor binding are indicated together with the distances observed in the Ag-HKT:INI structure. The chemical groups featuring the physiological substrate 3-HK are shown in light gray, and their interactions with the protein residues, resulting from manual modeling, are indicated. The arrow indicates the C4 position on the 4-(2-aminophenyl)-4-oxobutyric acid that would carry the proposed chemical modification consisting of the replacement of the carbonyl group by an oxy-amino (-O-NH₂) moiety.

Scheme 3.