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Abstract Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein‐derived cofactor. Reported herein is the discovery and elucidation of a Cys‐Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C−S) bond. By genetically incorporating an unnatural amino acid, 3,5‐difluoro‐tyrosine (F₂‐Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon–fluorine bond activation and fluoride release were identified by mass spectrometry and¹⁹F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low‐resolution techniques. Finally, a new sequence motif, C‐X‐Y‐Y(F), is proposed for identifying the Cys‐Tyr crosslink. 

Mononuclear, nonheme iron enzymes are known for their ability to mediate the oxidation of organic molecules in primary and secondary metabolism. One class of such enzymes is the diol dioxygenases that catalyze the oxidative cleavage of aromatic molecules. They come in two varieties, intradiol and extradiol, that add molecular oxygen symmetrically or asymmetrically, respectively. 3-Hydroxyanthranilate 3,4-dioxygenase (HAO) is a type III extradiol dioxygenase found in metabolic pathways related to breaking down tryptophan 2-nitrobenzoic acid. The product of HAO is unstable and either nonenzymatically cyclizes to quinolinic acid (QUIN), an endogenous neurotoxin and the universal precursor for NAD(P) biosynthesis, or is enzymatically processed, ultimately being fully oxidized to CO2 in the citric acid cycle. Elevation of QUIN is associated with neurodegenerative diseases, making HAO biomedically relevant. This article summarizes the history and current state of knowledge of the biochemistry of HAO. Recent studie that utilized X-ray crystallography of the in crystallo reactions coupled with various spectroscopies and activity measurements to elucidate much of the chemical mechanism catalyzed by HAO are highlighted. 

Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a non-heme iron center. It catalyzes oxidation of both cysteamine in sulfur metabolism and N-terminal cysteine-containing proteins or peptides, such as regulator of G protein signaling 5 (RGS5). It thereby preserves oxygen homeostasis in a variety of physiological processes. However, little is known about its catalytic center and how it interacts with these two types of primary substrates in addition to O2. Here, using EPR, Mössbauer, and UV-Vis spectroscopies, we explored the binding mode of cysteamine and RGS5 to human and mouse ADO proteins in their physiologically relevant ferrous form. This characterization revealed that in the presence of nitric oxide as a spin probe and oxygen surrogate, both the small molecule and the peptide substrates coordinate to the iron center with their free thiols in a monodentate binding mode, in sharp contrast to binding behaviors observed in other thiol dioxygenases. We observed a substrate-bound B-type dinitrosyl iron center complex in ADO, suggesting the possibility of dioxygen binding to the iron ion in a side-on mode. Moreover, we observed a substrate-mediated reduction of the ferric to the ferrous oxidation state at the iron center. Subsequent MS analysis indicated corresponding disulfide formation of the substrates, suggesting that the presence of the substrate could reactivate ADO to defend against oxidative stress. The findings of this work contribute to the understanding of the substrate interaction in ADO and fill a gap in our knowledge of the substrate specificity of thiol dioxygenases. 

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-resolvedin crystalloreactions 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. 

Fluorochemicals are a widely distributed class of compounds and have been utilized across a wide range of industries for decades. Given the environmental toxicity and adverse health threats of some fluorochemicals, the development of new methods for their decomposition is significant to public health. However, the carbon–fluorine (C–F) bond is among the most chemically robust bonds; consequently, the degradation of fluorinated hydrocarbons is exceptionally difficult. Here, metalloenzymes that catalyze the cleavage of this chemically challenging bond are reviewed. These enzymes include histidine-ligated heme-dependent dehaloperoxidase and tyrosine hydroxylase, thiolate-ligated heme-dependent cytochrome P450, and four nonheme oxygenases, namely, tetrahydrobiopterin-dependent aromatic amino acid hydroxylase, 2-oxoglutarate-dependent hydroxylase, Rieske dioxygenase, and thiol dioxygenase. While much of the literature regarding the aforementioned enzymes highlights their ability to catalyze C–H bond activation and functionalization, in many cases, the C–F bond cleavage has been shown to occur on fluorinated substrates. A copper-dependent laccase-mediated system representing an unnatural radical defluorination approach is also described. Detailed discussions on the structure–function relationships and catalytic mechanisms provide insights into biocatalytic defluorination, which may inspire drug design considerations and environmental remediation of halogenated contaminants.