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Parkinson's disease is the second most common neurodegenerative disorder, affecting nearly 10 million people worldwide. Ferroptosis, a recently identified form of regulated cell death characterized by 15-lipoxygenase-mediated hydroperoxidation of membrane lipids, has been implicated in neurodegenerative disorders including amyotrophic lateral sclerosis and Parkinson's disease. Pharmacological inhibition of 15 -lipoxygenase to prevent iron- and lipid peroxidation-associated ferroptotic cell death is a rational strategy for the treatment of Parkinson's disease. We report here the characterization of PTC-041 as an anti-ferroptotic reductive lipoxygenase inhibitor developed for the treatment of Parkinson's disease. In these studies, PTC-041 potently protects primary human Parkinson's disease patient-derived fibroblasts from lipid peroxidation and subsequent ferroptotic cell death and prevents ferroptosis-related neuronal loss and astrogliosis in primary rat neuronal cultures. Additionally, PTC-041 prevents ferroptotic-mediated α-synuclein protein aggregation and nitrosylation in vitro, suggesting a potential role for anti-ferroptotic lipoxygenase inhibitors in mitigating pathogenic aspects of synucleinopathies such as Parkinson's disease. We further found that PTC-041 protects against synucleinopathy in vivo, demonstrating that PTC-041 treatment of Line 61 transgenic mice protects against α-synuclein aggregation and phosphorylation as well as prevents associated neuronal and non-neuronal cell death. Finally, we show that. PTC-041 protects against 6-hydroxydopamine-induced motor deficits in a hemiparkinsonian rat model, further validating the potential therapeutic benefits of lipoxygenase inhibitors in the treatment of Parkinson's disease., Competing Interests: I have read the journal’s policy, and the authors of this manuscript have the following competing interests: AA, AM, JCL, KPM, MW, SO, JKT, and MBK are employees of PTC Therapeutics, Inc., a biotechnology company. In connection with such employment, the authors receive salary, benefits and stock-based compensation, including stock options, restricted stock, other stock-related grants, and the right to purchase discounted stock through PTC’s employee stock purchase plan. All other authors, RKW, declared no competing interests for this work. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Minnella et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ferroptosis is a form of programmed cell death associated with inflammation, neurodegeneration, and ischemia. Vitamin E (alpha-tocopherol) has been reported to prevent ferroptosis, but the mechanism by which this occurs is controversial. To elucidate the biochemical mechanism of vitamin E activity, we systematically investigated the effects of its major vitamers and metabolites on lipid oxidation and ferroptosis in a striatal cell model. We found that a specific endogenous metabolite of vitamin E, alpha-tocopherol hydroquinone, was a dramatically more potent inhibitor of ferroptosis than its parent compound, and inhibits 15-lipoxygenase via reduction of the enzyme's non-heme iron from its active Fe3+ state to an inactive Fe2+ state. Furthermore, a non-metabolizable isosteric analog of vitamin E which retains antioxidant activity neither inhibited 15-lipoxygenase nor prevented ferroptosis. These results call into question the prevailing model that vitamin E acts predominantly as a non-specific lipophilic antioxidant. We propose that, similar to the other lipophilic vitamins A, D and K, vitamin E is instead a pro-vitamin, with its quinone/hydroquinone metabolites responsible for its anti-ferroptotic cytoprotective activity., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: All authors are employees of, and/or hold equity in BioElectron Technology Corporation, Inc. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr use a shared radical-mediated mechanism. In addition to the radical SAM CX(3)CX(2)C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function, putatively to form (cysteine 355 in RlmN) and resolve (cysteine 118 in RlmN) a covalent intermediate needed to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further clarify the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species between RlmN and its rRNA substrate and well as the roles of the conserved cysteine residues in catalysis.

The continued ability to treat bacterial infections requires effective antibiotics. The development of new therapeutics is guided by knowledge of the mechanisms of action of and resistance to these antibiotics. Continued efforts to understand and counteract antibiotic resistance mechanisms at a molecular level have the potential to direct development of new therapeutic strategies in addition to providing insight into the underlying biochemical functions impacted by antibiotics. The interaction of antibiotics with the peptidyltransferase center and adjacent exit tunnel within the bacterial ribosome is the predominant mechanism by which antibiotics impede translation, thus stalling growth. Resistance enzymes catalyze the chemical modification of the RNA that composes these functional regions, leading to diminished binding of antibiotics. This review discusses recent advances in the elucidation of chemical mechanisms underlying resistance and driving the development of new antibiotics.

Enzymes that cleave C-H bonds are often found to depend on well-packed hydrophobic cores that influence the distance between the hydrogen donor and acceptor. Residue F159 in taurine alpha-ketoglutarate dioxygenase (TauD) is demonstrated to play an important role in the binding and orientation of its substrate, which undergoes a hydrogen atom transfer to the active site Fe(IV)=O. Mutation of F159 to smaller hydrophobic side chains (L, V, A) leads to substantially reduced rates for substrate binding and for C-H bond cleavage, as well as increased contribution of the chemical step to k(cat) under steady-state turnover conditions. The greater sensitivity of these substrate-dependent processes to mutation at position 159 than observed for the oxygen activation process supports a previous conclusion of modularity of function within the active site of TauD (McCusker, K. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 19791-19795). Extraction of intrinsic deuterium kinetic isotope effects (KIEs) using single turnover transients shows 2- to 4-fold increase in the size of the KIE for F159V in relation to wild-type and F159L. It appears that there is a break in behavior following removal of a single methylene from the side chain of F159L to generate F159V, whereby the protein active site loses its ability to restore the internuclear distance between substrate and Fe(IV)=O that supports optimal hydrogenic wave function overlap.

Taurine alpha-ketoglutarate dioxygenase (tauD) is one of the best-studied alpha-ketoglutarate (alphaKG)-dependent nonheme iron oxygenases. As with all oxygenases, a fine balance must be struck between generating a species sufficiently reactive for the required chemistry and controlling that species to prevent undesirable side reactions [Klinman JP (2007) Accts Chem Res 40:325-333]. In the case of tauD, the substrate oxidizing species has been shown to be a ferryl-oxo, and the introduction of deuterium at the reactive position of substrate results in an enormous kinetic isotope effect together with a partial uncoupling of oxygen activation from substrate oxidation [Price JC, Barr EW, Glass TE, Krebs C, Bollinger JM (2003) J Am Chem Soc 125:13008-13009]. We have generated a series of site-specific variants at a position that resides directly behind bound substrate (F159 to L, V, A, and G). Decreasing side-chain bulk diminishes the coupling of oxygen activation to C-H cleavage, which is further reduced by substrate deuteration. Despite this impact, oxygen activation remains completely coupled to the oxidative decarboxylation of alphaKG. The concentration of bis-Tris buffer impacts the extent of coupling of oxygen activation to C-H cleavage, implicating the buffer in the uncoupling pathway. These data indicate a critical role for residue 159 in substrate positioning and reaction in tauD and show that minor active-site perturbations in these enzymes could allow for changes in substrate reactivity while maintaining substrate triggering and oxygen binding/activation.

Contrasted here are the competitive 18O/16O kinetic isotope effects (18O KIEs) on kcat/Km(O2) for three non-heme iron enzymes that activate O2 at an iron center coordinated by a 2-His-1-carboxylate facial triad: taurine dioxygenase (TauD), (S)-(2)-hydroxypropylphosphonic acid epoxidase (HppE), and 1-aminocyclopropyl-1-carboxylic acid oxidase (ACCO). Measured 18O KIEs of 1.0102 +/- 0.0002 (TauD), 1.0120 +/- 0.0002 (HppE), and 1.0215 +/- 0.0005 (ACCO) suggest the formation in the rate-limiting step of O2 activation of an FeIII-peroxohemiketal, FeIII-OOH, and FeIV O species, respectively. The comparison of the measured 18O KIEs with calculated or experimental 18O equilibrium isotope effects (18O EIEs) provides new insights into the O2 activation through an inner-sphere mechanism at a non-heme iron center.

The behavior of three cyclic and three acyclic analogues of 1-aminocyclopropane-1-carboxylic acid (ACC) with ACC oxidase has been analyzed with regard to turnover rates, product distribution, and O(2) uncoupling. The cyclic analogues all form ethylene, and the acyclic analogues all undergo decarboxylation. The degree of uncoupling varies from almost none (ACC) to 21-fold (glycine), while turnover rates (k(cat)) are all within a factor of 4-fold of that of ACC. The aggregate data point toward a rate-determining formation of an activated iron-oxo intermediate, which partitions between amine oxidation and reductive uncoupling in a manner that is dependent on substrate structure.