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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.

SUBMITTER: Pidugu LS

PROVIDER: S-EPMC8493610 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mononuclear non-heme iron complexes that serve as structural and functional mimics of the thiol dioxygenases (TDOs), cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), have been prepared and characterized with crystallographic, spectroscopic, kinetic, and computational methods. The high-spin Fe(II) complexes feature the facially coordinating tris(4,5-diphenyl-1-methylimidazol-2-yl)phosphine (Ph2TIP) ligand that replicates the three histidine (3His) triad of the TDO active sites. Further coordination with bidentate l-cysteine ethyl ester (CysOEt) or cysteamine (CysAm) anions yielded five-coordinate (5C) complexes that resemble the substrate-bound forms of CDO and ADO, respectively. Detailed electronic-structure descriptions of the [Fe(Ph2TIP)(LS,N)]BPh4 complexes, where LS,N = CysOEt (1) or CysAm (2), were generated through a combination of spectroscopic techniques [electronic absorption, magnetic circular dichroism (MCD)] and density functional theory (DFT). Complexes 1 and 2 decompose in the presence of O2 to yield the corresponding sulfinic acid (RSO2H) products, thereby emulating the reactivity of the TDO enzymes and related complexes. Rate constants and activation parameters for the dioxygenation reactions were measured and interpreted with the aid of DFT calculations for O2-bound intermediates. Treatment of the TDO models with nitric oxide (NO)-a well-established surrogate of O2-led to a mixture of high-spin and low-spin {FeNO}7 species at low temperature (-70 °C), as indicated by electron paramagnetic resonance (EPR) spectroscopy. At room temperature, these Fe/NO adducts convert to a common species with EPR and infrared (IR) features typical of cationic dinitrosyl iron complexes (DNICs). To complement these results, parallel spectroscopic, computational, and O2/NO reactivity studies were carried out using previously reported TDO models that feature an anionic hydrotris(3-phenyl-5-methyl-pyrazolyl)borate (Ph,MeTp-) ligand. Though the O2 reactivities of the Ph2TIP- and Ph,MeTp-based complexes are quite similar, the supporting ligand perturbs the energies of Fe 3d-based molecular orbitals and modulates Fe-S bond covalency, suggesting possible rationales for the presence of neutral 3His coordination in CDO and ADO.

Dataset Information

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

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