Crystal structures of human 3-hydroxyanthranilate 3,4-dioxygenase with native and non-native metals bound in the active site

Abstract

3-Hydroxyanthranilate 3,4-dioxygenase (3HAO) is an enzyme in the microglial branch of the kynurenine pathway of tryptophan degradation. 3HAO is a non-heme iron-containing, ring-cleaving extradiol dioxygenase that catalyzes the addition of both atoms of O₂ to the kynurenine pathway metabolite 3-hydroxyanthranilic acid (3-HANA) to form quinolinic acid (QUIN). QUIN is a highly potent excitotoxin that has been implicated in a number of neurodegenerative conditions, making 3HAO a target for pharmacological downregulation. Here, the first crystal structure of human 3HAO with the native iron bound in its active site is presented, together with an additional structure with zinc (a known inhibitor of human 3HAO) bound in the active site. The metal-binding environment is examined both structurally and via inductively coupled plasma mass spectrometry (ICP-MS), X-ray fluorescence spectroscopy (XRF) and electron paramagnetic resonance spectroscopy (EPR). The studies identified Met35 as the source of potential new interactions with substrates and inhibitors, which may prove useful in future therapeutic efforts.

Keywords: 3-hydroxyanthranilate 3,4-dioxygenase; kynurenine pathway; neurodegeneration; oxidoreductase; quinolinic acid.

Figure 1

The major reactions that occur via the kynurenine pathway of tryptophan degradation. The enzyme 3-­hydroxyanthranilic acid dioxygenase (3HAO, red) generates the potent excitotoxin quinolinic acid (QUIN, red box) as part of the microglial branch of the kynurenine pathway.

Figure 2

The overall structure of human 3HAO (a) compared with R. metallodurans 3HAO (b). Fe atoms are shown as orange spheres. The cupin domain of human 3HAO (red) has the same fold as R. metallodurans 3HAO (dark red). Human 3HAO contains an additional domain (blue) which contains a core jelly-roll motif. The function of this domain is unknown.

Figure 3

Coordination of iron (left, orange sphere) and zinc (right, gray sphere) in the h3HAO active site. In each, His46 and His91 contribute one nitrogen ligand, Glu53 forms a bidentate interaction and two tightly bound water molecules complete the coordination sphere. Metal–ligand interactions are indicated by light blue dashed lines.

Figure 4

X-ray fluorescence spectrum of an Fe(III)-h3HAO crystal. Overlay of the spectrum of the crystal (blue) versus the blank (red) clearly shows the presence of Fe Kα and Kβ peaks. No other metals were detected. Peaks for Si and Ar are present owing to the glass capillary and the air around the sample, respectively.

Figure 5

EPR spectra of air-free (black) and air-exposed (red) Fe(II)-h3HAO. Spectra were collected at a temperature of 12 K with a microwave frequency of 9.43 GHz, a microwave power of 20 mW, a modulation amplitude of 8 G and a sweep time of 41.9 s. The lack of signal in the anaerobic sample and the peak centered at g = 4.3 are indicative of the expected d ⁶ and d ⁵ electronic configurations.

Figure 6

Comparison of the metal–ligand distances for all of the available mammalian 3HAO crystal structures: Fe(III)-h3HAO (this work; PDB entry 5tk5), Zn(II)-h3HAO (this work; PDB entry 5tkq), Ni(II)-h3HAO (PDB entry 2qnk) and bovine 3HAO [Fe(III)-Bt3HAO; Đilović et al., 2009 ▸]. Fe atoms are shown as orange spheres, the Ni atom is shown as a green sphere and the Zn atom is shown as a gray sphere. Metal–ligand interactions are indicated by light blue dashed lines.

Figure 7

Superposition of Fe(III)-h3HAO (cyan) and R. metallodurans 3HAO (red). Fe atoms are shown as orange spheres. The N- and C-termini of Fe(III)-h3HAO are labeled. The C-terminus of R. metallodurans 3HAO is labeled with a red asterisk. The locations of both the rubredoxin-like FeS₄ center and helix α3 in R. metallodurans 3HAO are indicated.

Figure 8

Superposition of key active-site residues of Fe(III)-h3HAO (green C atoms) and R. metallodurans 3HAO (gray C atoms) in complex with the inhibitor 4-Cl-3-HANA (magenta C atoms). Fe atoms are shown as orange spheres. (a) The active sites superimposed showing that all of the residues implicated in inhibitor binding are structurally well conserved, with the exception that Arg47 in R. metallodurans 3HAO, which interacts with bound oxygen in the complex (not shown), shifts relative to its Fe(III)-h3HAO counterpart Arg43. (b) The same view but with the nonconserved Met35 shown in comparison to its R. metallodurans 3HAO counterpart Thr39. Met35 projects into the active site near, but not close enough to interact with, a bound ligand. However, a minor reorientation of Met35 could result in a van der Waals interaction with a substrate or inhibitor.