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

   https://doi.org/10.1073/pnas.2005327117  

   Wang, Yifan ; Liu, Kathy Fange ; Yang, Yu ; Davis, Ian ; Liu, Aimin ( July 2020 , Proceedings of the National Academy of Sciences)

   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.
2. OvoA _Mtht from Methyloversatilis thermotolerans ovothiol biosynthesis is a bifunction enzyme: thiol oxygenase and sulfoxide synthase activities

   https://doi.org/10.1039/D1SC05479A  

   Cheng, Ronghai ; Weitz, Andrew C. ; Paris, Jared ; Tang, Yijie ; Zhang, Jingyu ; Song, Heng ; Naowarojna, Nathchar ; Li, Kelin ; Qiao, Lu ; Lopez, Juan ; et al ( March 2022 , Chemical Science)

   Mononuclear non-heme iron enzymes are a large class of enzymes catalyzing a wide-range of reactions. In this work, we report that a non-heme iron enzyme in Methyloversatilis thermotolerans , OvoA Mtht, has two different activities, as a thiol oxygenase and a sulfoxide synthase. When cysteine is presented as the only substrate, OvoA Mtht is a thiol oxygenase. In the presence of both histidine and cysteine as substrates, OvoA Mtht catalyzes the oxidative coupling between histidine and cysteine (a sulfoxide synthase). Additionally, we demonstrate that both substrates and the active site iron's secondary coordination shell residues exert exquisite control over the dual activities of OvoA Mtht (sulfoxide synthase vs. thiol oxygenase activities). OvoA Mtht is an excellent system for future detailed mechanistic investigation on how metal ligands and secondary coordination shell residues fine-tune the iron-center electronic properties to achieve different reactivities.
3. Trapping a cross-linked lysine–tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB

   https://doi.org/10.1073/pnas.2101571118  

   Balo, Aidin R. ; Caruso, Alessio ; Tao, Lizhi ; Tantillo, Dean J. ; Seyedsayamdost, Mohammad R. ; Britt, R. David ( May 2021 , Proceedings of the National Academy of Sciences)

   The radicalS-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon–carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys–Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]²⁺state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys–Trp radical (Lys–Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys–Trp• intermediate by the redox-tuned [4Fe-4S]²⁺AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon–carbon bond-forming reactions.
4. Characterization of the nickel-inserting cyclometallase LarC from Moorella thermoacetica and identification of a cytidinylylated reaction intermediate

   https://doi.org/10.1093/mtomcs/mfac014  

   Turmo, Aiko ; Hu, Jian ; Hausinger, Robert P. ( February 2022 , Metallomics)

   LarC catalyzes the CTP-dependent insertion of nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide (P2TMN), the final biosynthetic step for generating the nickel-pincer nucleotide (NPN) enzyme cofactor. In this study, we characterized a LarC homolog from Moorella thermoacetica (LarCMt) and characterized selected properties of the protein. We ruled out the hypothesis that enzyme inhibition by its product pyrophosphate accounts for its apparent single-turnover activity. Most notably, we identified a cytidinylylated-substrate intermediate that is formed during the reaction of LarCMt. Selected LarCMt variants with substitutions at the predicted CTP-binding site retained substantial amounts of activity, but exhibited greatly reduced levels of the CMP-P2TMN intermediate. In contrast, enhanced amounts of the CMP-P2TMN intermediate were generated when using LarCMt from cells grown on medium without supplemental nickel. On the basis of these results, we propose a functional role for CTP in the unprecedented nickel-insertase reaction during NPN biosynthesis.
5. Hydrogen Movements in the Oxidative Half-Reaction of Kynurenine 3-Monooxygenase from Pseudomonas fluorescens Reveal the Mechanism of Hydroxylation

   Brett A. Beaupre, Karen R. ( July 2020 , Archives of biochemistry and biophysics)

   Kynurenine 3-monoxygenase (KMO) catalyzes the conversion of L-kynurenine (L-Kyn) to 3-hydroxykynurenine (3-OHKyn) in the pathway for tryptophan catabolism. We have investigated the effects of pH and deuterium substitution on the oxidative half-reaction of KMO from P. fluorescens (PfKMO). The three phases observed during the oxidative half reaction are formation of the hydroperoxyflavin, hydroxylation and product release. The measured rate constants for these phases proved largely unchanging with pH, suggesting that the KMO active site is insulated from exchange with solvent during catalysis. A solvent inventory study indicated that a solvent isotope effect of 2 - 3 is observed for the hydroxylation phase and that two or more protons are in flight during this step. An inverse isotope effect of 0.84  0.01 on the rate constant for the hydroxylation step with ring perdeutero-L-Kyn as a substrate indicates a shift from sp2 to sp3 hybridization in the transition state leading to the formation of a non-aromatic intermediate. The pH dependence of transient state data collected for the substrate analog meta-nitrobenzoylalanine indicate that groups proximal to the hydroperoxyflavin are titrated in the range pH 5 - 8.5 and can be described by a pKa of 8.8. That the higher pH values domore »not slow the rate of hydroxylation precludes that the pKa measured pertains to the proton of the hydroperoxflavin. Together, these observations indicate that the C4a-hydroperoxyflavin has a pKa >> 8.5, that a non-aromatic species is the immediate product of hydroxylation and that at least two solvent derived protons are in-flight during oxygen insertion to the substrate aromatic ring. A unifying mechanistic proposal for these observations is proposed.« less