Observing 3-hydroxyanthranilate-3,4-dioxygenase in action through a crystalline lens

Abstract

The synthesis of quinolinic acid from tryptophan is a critical step in the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+) in mammals. Herein, the nonheme iron-based 3-hydroxyanthranilate-3,4-dioxygenase responsible for quinolinic acid production was studied by performing time-resolved in crystallo reactions monitored by UV-vis microspectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and X-ray crystallography. Seven catalytic intermediates were kinetically and structurally resolved in the crystalline state, and each accompanies protein conformational changes at the active site. Among them, a monooxygenated, seven-membered lactone intermediate as a monodentate ligand of the iron center at 1.59-Å resolution was captured, which presumably corresponds to a substrate-based radical species observed by EPR using a slurry of small-sized single crystals. Other structural snapshots determined at around 2.0-Å resolution include monodentate and subsequently bidentate coordinated substrate, superoxo, alkylperoxo, and two metal-bound enol tautomers of the unstable dioxygenase product. These results reveal a detailed stepwise O-atom transfer dioxygenase mechanism along with potential isomerization activity that fine-tunes product profiling and affects the production of quinolinic acid at a junction of the metabolic pathway.

Molecular Rationale for Partitioning between C–H and C–F Bond Activation in Heme-Dependent Tyrosine Hydroxylase

“…As shown in Figure A (black trace), the 3-F-Tyr-bound binary complex crystal showed a Soret peak maximum at 400 nm and other features at 499, 532, 570, 626 nm, which were in line with absorption spectra obtained in the solution state. The slight differences of λ max between crystalline and solution states are anticipated, resulting from crystal packing and pH difference as reported in a nonheme dioxygenase study . In addition, the absorbance of Soret peaks was easily saturated due to the dense packing of crystals, hence the readings of λ max based on the peak shape could introduce some inaccuracy.…”

Crystal structure of human cysteamine dioxygenase provides a structural rationale for its function as an oxygen sensor

“…Proline is known as a helix breaker. However, a proline–proline pair does not interrupt helices, as we have previously found in another nonheme Fe dioxygenase ( 42 , 49 ). It is known that peptide bond synthesis is particularly slow with proline involved, and the production of two adjacent prolines often results in ribosome stalling ( 50 , 51 , 52 ).…”

“…Both ends are accessible to solvent. Presumably, the front end with Loops 6 and 8 binds the primary substrate, while the back end with Loop 5 is reserved for oxygen binding, as we recently found in a nonheme iron dioxygenase with a cupin structure ( 42 ).…”

Microbial paracetamol degradation involves a high diversity of novel amidase enzyme candidates

“…The ring cleavage of aromatic compounds containing hydroxyl and amino substituents (i.e. 2-aminophenol, 3-hydroxyanthranilate, and 5-aminosalicylate) has been previously reported (Hintner et al, 2004; Li de et al, 2013; Takenaka et al, 1997; Wang et al, 2020). Therefore, the aromatic ring of 4-AP might also be cleaved before deamination by class III ring-cleaving dioxygenases.…”