Structural, Spectroscopic, and Computational Insights from Canavanine-Bound and Two Catalytically Compromised Variants of the Ethylene-Forming Enzyme.

The ethylene-forming enzyme (EFE) is an Fe(II), 2-oxoglutarate (2OG), and l-arginine (l-Arg)-dependent oxygenase that either forms ethylene and three CO2/bicarbonate from 2OG or couples the decarboxylation of 2OG to C5 hydroxylation of l-Arg. l-Arg binds with C5 toward the metal center, causing 2OG to change from monodentate to chelate metal interaction and OD1 to OD2 switch of D191 metal coordination. We applied anaerobic UV-visible spectroscopy, X-ray crystallography, and computational approaches to three EFE systems with high-resolution structures. The ineffective l-Arg analogue l-canavanine binds to the EFE with O5 pointing away from the metal center while promoting chelate formation by 2OG but fails to switch the D191 metal coordination from OD1 to OD2. Substituting alanine for R171 that interacts with 2OG and l-Arg inactivates the protein, prevents metal chelation by 2OG, and weakens l-Arg binding. The R171A EFE had electron density at the 2OG binding site that was identified by mass spectrometry as benzoic acid. The substitution by alanine of Y306 in the EFE, a residue 12 Å away from the catalytic metal center, generates an interior cavity that leads to multiple local and distal structural changes that reduce l-Arg binding and significantly reduce the enzyme activity. Flexibility analyses revealed correlated and anticorrelated motions in each system, with important distinctions from the wild-type enzyme. In combination, the results are congruent with the currently proposed enzyme mechanism, reinforce the importance of metal coordination by OD2 of D191, and highlight the importance of the second coordination sphere and longer range interactions in promoting EFE activity.

Mitochondrial transcription factor A (TFAM) has 5'-deoxyribose phosphate lyase activity in vitro.

Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER, one of the toxic repair intermediates is 5'-deoxyribose phosphate (5'dRp). Human mitochondrial DNA polymerase γ has weak dRp lyase activities, and another known dRp lyase in the nucleus, human DNA polymerase ß, can also localize to mitochondria in certain cell and tissue types. Nonetheless, whether additional proteins have the ability to remove 5'dRp in mitochondria remains unknown. Our prior work on the AP lyase activity of mitochondrial transcription factor A (TFAM) has prompted us to examine its ability to remove 5'dRp residues in vitro. TFAM is the primary DNA-packaging factor in human mitochondria and interacts with mitochondrial DNA extensively. Our data demonstrate that TFAM has the dRp lyase activity with different DNA substrates. Under single-turnover conditions, TFAM removes 5'dRp residues at a rate comparable to that of DNA polymerase (pol) ß, albeit slower than that of pol λ. Among the three proteins examined, pol λ shows the highest single-turnover rates in dRp lyase reactions. The catalytic effect of TFAM is facilitated by lysine residues of TFAM via Schiff base chemistry, as evidenced by the observation of dRp-lysine adducts in mass spectrometry experiments. The catalytic effect of TFAM observed here is analogous to the AP lyase activity of TFAM reported previously. Together, these results suggest a potential role of TFAM in preventing the accumulation of toxic DNA repair intermediates.

Identification of Key Ubiquitination Sites Involved in the Proteasomal Degradation of AtACS7 in Arabidopsis.

The gaseous hormone ethylene plays pivotal roles in plant growth and development. The rate-limiting enzyme of ethylene biosynthesis in seed plants is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). ACS proteins are encoded by a multigene family and the expression of ACS genes is highly regulated, especially at a post-translational level. AtACS7, the only type III ACS in Arabidopsis, is degraded in a 26S proteasome-dependent pathway. Here, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, two lysine residues of AtACS7, lys285 (K285) and lys366 (K366), were revealed to be ubiquitin-modified in young, light-grown Arabidopsis seedlings but not in etiolated seedlings. Deubiquitylation-mimicking mutations of these residues significantly increased the stability of the AtACS7K285RK366R mutant protein in cell-free degradation assays. All results suggest that K285 and K366 are the major ubiquitination sites on AtACS7, providing deeper insights into the post-translational regulation of AtACS7 in Arabidopsis.

Systematic Analysis of the MIO-forming Residues of Aromatic Ammonia Lyases.

Aromatic ammonia lyases (AALs) and tyrosine/phenylalanine ammonia mutases (TAM/PAM) are 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent enzymes. Usually, the MIO moiety is autocatalytically formed from the tripeptide Ala-Ser-Gly (ASG) and acts as an electrophile during the enzymatic reaction. However, the MIO-forming residues (ASG) have some diversity in this enzyme class. In this work, a systematic investigation on the variety of MIO-forming residues was carried out using in-depth sequence analyses. Several protein clusters of AAL-like enzymes with unusual MIO-forming residues such as ACG, TSG, SSG, and CSG were identified, including two novel histidine ammonia lyases and one PAM with CSG and TSG residues, respectively, as well as three novel ergothioneine trimethylammonia lyases without MIO motif. The mutagenesis of common MIO-groups confirmed the function of these MIO variants, which provides good starting points for future functional prediction and mutagenesis research of AALs.

Review on sterilization techniques, and the application potential of phage lyase and lyase immobilization in fighting drug-resistant bacteria.

Many human health problems and property losses caused by pathogenic contamination cannot be underestimated. Bactericidal techniques have been extensively studied to address this issue of public health and economy. Bacterial resistance develops as a result of the extensive use of single or multiple but persistent usage of sterilizing drugs, and the emergence of super-resistant bacteria brings new challenges. Therefore, it is crucial to control pathogen contamination by applying innovative and effective sterilization techniques. As organisms that exist in nature and can specifically kill bacteria, phages have become the focus as an alternative to antibacterial agents. Furthermore, phage-encoded lyases are proteins that play important roles in phage sterilization. The in vitro sterilization of phage lyase has been developed as a novel biosterilization technique to reduce bacterial resistance and is more environmentally friendly than conventional sterilization treatments. For the shortcomings of enzyme applications, this review discusses the enzyme immobilization methods and the application potential of immobilized lyases for sterilization. Although some techniques provide effective solutions, immobilized lyase sterilization technology has been proven to be a more effective innovation for efficient pathogen killing and reducing bacterial resistance. We hope that this review can provide new insights for the development of sterilization techniques.

Charge neutralization and ß-elimination cleavage mechanism of family 42 L-rhamnose-α-1,4-D-glucuronate lyase revealed using neutron crystallography.

Gum arabic (GA) is widely used as an emulsion stabilizer and edible coating and consists of a complex carbohydrate moiety with a rhamnosyl-glucuronate group capping the non-reducing ends. Enzymes that can specifically cleave the glycosidic chains of GA and modify their properties are valuable for structural analysis and industrial application. Cryogenic X-ray crystal structure of GA-specific L-rhamnose-α-1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1), belonging to the polysaccharide lyase (PL) family 42, has been previously reported. To determine the specific reaction mechanism based on its hydrogen-containing enzyme structure, we performed joint X-ray/neutron crystallography of FoRham1. Large crystals were grown in the presence of L-rhamnose (a reaction product), and neutron and X-ray diffraction datasets were collected at room temperature at 1.80 and 1.25 Å resolutions, respectively. The active site contained L-rhamnose and acetate, the latter being a partial analog of glucuronate. Incomplete H/D exchange between Arg166 and acetate suggested that a strong salt-bridge interaction was maintained. Doubly deuterated His105 and deuterated Tyr150 supported the interaction between Arg166 and the acetate. The unique hydrogen-rich environment functions as a charge neutralizer for glucuronate and stabilizes the oxyanion intermediate. The NE2 atom of His85 was deprotonated and formed a hydrogen bond with the deuterated O1 hydroxy of L-rhamnose, indicating the function of His85 as the base/acid catalyst for bond cleavage via ß-elimination. Asp83 functions as a pivot between the two catalytic histidine residues by bridging them. This His-His-Asp structural motif is conserved in the PL 24, 25, and 42 families.

GT-11 impairs insulin signaling through modulation of sphingolipid metabolism in C2C12 myotubes.

AIMS: Sphingolipids are involved in the regulation of insulin signaling, which is linked to the development of insulin resistance, leading to diabetes mellitus. We aimed to study whether modulation of sphingolipid levels by GT-11 may regulate insulin signaling in C2C12 myotubes. MAIN METHODS: We investigated the effects of sphingolipid metabolism on Akt phosphorylation and glucose uptake using C2C12 myotubes. Either GT-11, an inhibitor of dihydroceramide desaturase 1 and S1P lyase, or siRNA targeting Sgpl1, the gene encoding the enzyme, was employed to determine the effect of sphingolipid metabolism modulation on insulin signaling. Western blotting and glucose uptake assays were used to evaluate the effect of treatments on insulin signaling. Sphingolipid metabolites were analyzed by high performance liquid chromatography (HPLC). KEY FINDINGS: Treatment with GT-11 resulted in decreased Akt phosphorylation and reduced glucose uptake. Silencing the Sgpl1 gene, which encodes S1P lyase, mimicked these findings, suggesting the potential for regulating insulin signaling through S1P lyase modulation. GT-11 modulated sphingolipid metabolism, inducing the accumulation of sphingolipids. Using PF-543 and ARN14974 to inhibit sphingosine kinases and acid ceramidase, respectively, we identified a significant interplay between sphingosine, S1P lyase, and insulin signaling. Treatment with either exogenous sphingosine or palmitic acid inhibited Akt phosphorylation, and reduced S1P lyase activity. SIGNIFICANCE: Our findings highlight the importance of close relationship between sphingolipid metabolism and insulin signaling in C2C12 myotubes, pointing to its potential therapeutic relevance for diabetes mellitus.

Gasotransmitters do not prevent changes in transepithelial ion transport induced by hypoxia followed by reoxygenation.

OBJECTIVES: How gaseous signalling molecules affect ion transport processes contributing to the physiological functions of the gastrointestinal tract under hypoxic conditions still needs to be clarified. The objective of the present study was to characterize the impact of gaseous signalling molecules on parameters of colonic ion transport during a hypoxia/reoxygenation cycle and the remaining secretory capacity of the epithelium after such a cycle. METHODS: Short-circuit current (Isc) and tissue conductance (Gt) recordings in Ussing chamber experiments were performed on rat colon samples using CORM-2 (putative CO donor; 35 and 350 µM), sodium nitroprusside (NO donor; 100 µM), NaHS (fast H2S donor; 10 - 1,000 µM), GYY 4137 (slow H2S donor; 50 µM) and Angeli's salt (HNO donor; 100 µM) as donors for gasotransmitters. Inhibition of endogenous synthesis of H2S was operated by inhibitors of cystathionin-γ-lyase, i.e. dl-propargylglycine (1 mM) or ß-cyano-l-alanine (5 mM), and the inhibitor of cystathionine-ß-synthase, amino-oxyacetate (5 mM). RESULTS: The fast gasotransmitter donors NaHS, sodium nitroprusside and Angeli's salt, administered 5 min before the onset of hypoxia, induced an increase in Isc. The response to the subsequently applied hypoxia was characterized by a decrease in Isc, which tended to be reduced only in the presence of the lowest concentration of NaHS (10 µM) tested. Reoxygenation resulted in a slow increase in Isc, which was unaffected by all donors or inhibitors tested. The stable acetylcholine derivative carbachol (50 µM) was administered at the end of each hypoxia/reoxygenation cycle to test the secretory capacity of the epithelium. Pretreatment of the tissue with the putative CO donor CORM-2 suppressed the secretory response induced by carbachol. The same was observed when cystathionin-γ-lyase and cystathionin-γ-synthase were inhibited simultaneously. Under both conditions, Gt drastically increased suggesting an impaired tissue integrity. CONCLUSIONS: The present results demonstrate that none of the exogenous gasotransmitter releasing drugs significantly ameliorated the changes in epithelial ion transport during the hypoxia/reoxygenation cycle ex vivo. In contrast, the putative CO donor CORM-2 exerted a toxic effect on the epithelium. The endogenous production of H2S, however, seems to have a protective effect on the mucosal integrity and the epithelial transport functions, which - when inhibited - leads to a loss of the secretory ability of the mucosa. This observation together with the trend for improvement observed with a low concentration of the H2S donor NaHS suggests a moderate protective role of low concentrations of H2S under hypoxic conditions.

The Ethylene Biosynthetic Enzymes, 1-Aminocyclopropane-1-Carboxylate (ACC) Synthase (ACS) and ACC Oxidase (ACO): The Less Explored Players in Abiotic Stress Tolerance.

Ethylene is an essential plant hormone, critical in various physiological processes. These processes include seed germination, leaf senescence, fruit ripening, and the plant's response to environmental stressors. Ethylene biosynthesis is tightly regulated by two key enzymes, namely 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO). Initially, the prevailing hypothesis suggested that ACS is the limiting factor in the ethylene biosynthesis pathway. Nevertheless, accumulating evidence from various studies has demonstrated that ACO, under specific circumstances, acts as the rate-limiting enzyme in ethylene production. Under normal developmental processes, ACS and ACO collaborate to maintain balanced ethylene production, ensuring proper plant growth and physiology. However, under abiotic stress conditions, such as drought, salinity, extreme temperatures, or pathogen attack, the regulation of ethylene biosynthesis becomes critical for plants' survival. This review highlights the structural characteristics and examines the transcriptional, post-transcriptional, and post-translational regulation of ACS and ACO and their role under abiotic stress conditions. Reviews on the role of ethylene signaling in abiotic stress adaptation are available. However, a review delineating the role of ACS and ACO in abiotic stress acclimation is unavailable. Exploring how particular ACS and ACO isoforms contribute to a specific plant's response to various abiotic stresses and understanding how they are regulated can guide the development of focused strategies. These strategies aim to enhance a plant's ability to cope with environmental challenges more effectively.

ACC SYNTHASE4 inhibits gibberellin biosynthesis and FLOWERING LOCUS T expression during citrus flowering.

Flowering is an essential process in fruit trees. Flower number and timing have a substantial impact on the yield and maturity of fruit. Ethylene and gibberellin (GA) play vital roles in flowering, but the mechanism of coordinated regulation of flowering in woody plants by GA and ethylene is still unclear. In this study, a lemon (Citrus limon L. Burm) 1-aminocyclopropane-1-carboxylic acid synthase gene (CiACS4) was overexpressed in Nicotiana tabacum and resulted in late flowering and increased flower number. Further transformation of citrus revealed that ethylene and starch content increased, and soluble sugar content decreased in 35S:CiACS4 lemon. Inhibition of CiACS4 in lemon resulted in effects opposite to that of 35S:CiACS4 in transgenic plants. Overexpression of the CiACS4-interacting protein ETHYLENE RESPONSE FACTOR3 (CiERF3) in N. tabacum resulted in delayed flowering and more flowers. Further experiments revealed that the CiACS4-CiERF3 complex can bind the promoters of FLOWERING LOCUS T (CiFT) and GOLDEN2-LIKE (CiFE) and suppress their expression. Moreover, overexpression of CiFE in N. tabacum led to early flowering and decreased flowers, and ethylene, starch, and soluble sugar contents were opposite to those in 35S:CiACS4 transgenic plants. Interestingly, CiFE also bound the promoter of CiFT. Additionally, GA3 and 1-aminocyclopropanecarboxylic acid (ACC) treatments delayed flowering in adult citrus, and treatment with GA and ethylene inhibitors increased flower number. ACC treatment also inhibited the expression of CiFT and CiFE. This study provides a theoretical basis for the application of ethylene to regulate flower number and mitigate the impacts of extreme weather on citrus yield due to delayed flowering.

Steric Perturbation of the Grob-like Final Step of Ethylene-Forming Enzyme Enables 3-Hydroxypropionate and Propylene Production.

Ethylene-forming enzyme (EFE) is an iron(II)-dependent dioxygenase that fragments 2-oxoglutarate (2OG) to ethylene (from C3 and C4) and 3 equivs of carbon dioxide (from C1, C2, and C5). This major ethylene-forming pathway requires l-arginine as the effector and competes with a minor pathway that merely decarboxylates 2OG to succinate as it oxidatively fragments l-arginine. We previously proposed that ethylene forms in a polar-concerted (Grob-like) fragmentation of a (2-carboxyethyl)carbonatoiron(II) intermediate, formed by the coupling of a C3-C5-derived propion-3-yl radical to a C1-derived carbonate coordinated to the Fe(III) cofactor. Replacement of one or both C4 hydrogens of 2OG by fluorine, methyl, or hydroxyl favored the elimination products 2-(F1-2/Me/OH)-3-hydroxypropionate and CO2 over the expected olefin or carbonyl products, implying strict stereoelectronic requirements in the final step, as is known for Grob fragmentations. Here, we substituted active-site residues expected to interact sterically with the proposed Grob intermediate, aiming to disrupt or enable the antiperiplanar disposition of the carboxylate electrofuge and carbonate nucleofuge required for concerted fragmentation. The bulk-increasing A198L substitution barely affects the first partition between the major and minor pathways but then, as intended, markedly diminishes ethylene production in favor of 3-hydroxypropionate. Conversely, the bulk-diminishing L206V substitution enables propylene formation from (4R)-methyl-2OG, presumably by allowing the otherwise sterically disfavored antiperiplanar conformation of the Grob intermediate bearing the extra methyl group. The results provide additional evidence for a polar-concerted ethylene-yielding step and thus for the proposed radical-polar crossover via substrate-radical coupling to the Fe(III)-coordinated carbonate.

Metabolic modulation of histone acetylation mediated by HMGCL activates the FOXM1/ß-catenin pathway in glioblastoma.

BACKGROUND: Altered branched-chain amino acid (BCAA) metabolism modulates epigenetic modification, such as H3K27ac in cancer, thus providing a link between metabolic reprogramming and epigenetic change, which are prominent hallmarks of glioblastoma multiforme (GBM). Here, we identified mitochondrial 3-hydroxymethyl-3-methylglutaryl-CoA lyase (HMGCL), an enzyme involved in leucine degradation, promoting GBM progression and glioma stem cell (GSC) maintenance. METHODS: In silico analysis was performed to identify specific molecules involved in multiple processes. Glioblastoma multiforme cells were infected with knockdown/overexpression lentiviral constructs of HMGCL to assess malignant performance in vitro and in an orthotopic xenograft model. RNA sequencing was used to identify potential downstream molecular targets. RESULTS: HMGCL, as a gene, increased in GBM and was associated with poor survival in patients. Knockdown of HMGCL suppressed proliferation and invasion in vitro and in vivo. Acetyl-CoA was decreased with HMGCL knockdown, which led to reduced NFAT1 nuclear accumulation and H3K27ac level. RNA sequencing-based transcriptomic profiling revealed FOXM1 as a candidate downstream target, and HMGCL-mediated H3K27ac modification in the FOXM1 promoter induced transcription of the gene. Loss of FOXM1 protein with HMGCL knockdown led to decreased nuclear translocation and thus activity of ß-catenin, a known oncogene. Finally, JIB-04, a small molecule confirmed to bind to HMGCL, suppressed GBM tumorigenesis in vitro and in vivo. CONCLUSIONS: Changes in acetyl-CoA levels induced by HMGCL altered H3K27ac modification, which triggers transcription of FOXM1 and ß-catenin nuclear translocation. Targeting HMGCL by JIB-04 inhibited tumor growth, indicating that mediators of BCAA metabolism may serve as molecular targets for effective GBM treatment.

Structure of mycobacterial ergothioneine-biosynthesis C-S lyase EgtE.

L-ergothioneine is widely distributed among various microbes to regulate their physiology and pathogenicity within complex environments. One of the key steps in the ergothioneine-biosynthesis pathway, the C-S bond cleavage reaction, uses the pyridoxal 5'-phosphate dependent C-S lyase to produce the final product L-ergothioneine. Here, we present the crystallographic structure of the ergothioneine-biosynthesis C-S lyase EgtE from Mycobacterium smegmatis (MsEgtE) represents the first published structure of ergothioneine-biosynthesis C-S lyases in bacteria and shows the effects of active site residues on the enzymatic reaction. The MsEgtE and the previously reported ergothioneine-biosynthesis C-S lyase Egt2 from Neurospora crassa (NcEgt2) fold similarly. However, discrepancies arise in terms of substrate recognition, as observed through sequence and structure comparison of MsEgtE and NcEgt2. The structural-based sequence alignment of the ergothioneine-biosynthesis C-S lyase from fungi and bacteria shows clear distinctions among the recognized substrate residues, but Arg348 is critical and an extremely conserved residue for substrate recognition. The α14 helix is exclusively found in the bacteria EgtE, which represent the most significant difference between bacteria EgtE and fungi Egt2, possibly resulting from the convergent evolution of bacteria and fungi.

Exogenous C-S Lyase Enzyme, a Potential Tool To Release Aromas in Wine or Beer?

Varietal thiols have an impact on the overall aroma of many white, rosé, and red wines and beers. They originate from the metabolism of non-odorant aroma precursors by yeast during the fermentation step, via an intrinsic enzyme, the carbon-sulfur ß-lyase (CSL, EC 4.4.1.13). However, this metabolism is directly dependent upon efficient internalization of aroma precursors and intracellular CSL activity. Consequently, the overall CSL activity converts on average only 1% of the total precursors available. To improve the conversion of thiol precursors during winemaking or brewing, we investigated the possibility of using an exogenous CSL enzyme from Lactobacillus delbrueckii subsp. bulgaricus produced in Escherichia coli. We first implemented a reliable spectrophotometric method to monitor its activity on different related aroma precursors and studied its activity in the presence of various competing analogues and at different pH values. This study allowed us to highlight the parameters to define CSL activity and structural insights for the recognition of the substrate, which pave the way for the use of exogenous CSL for the release of aromas in beer and wine.

A key mutation in magnesium chelatase I subunit leads to a chlorophyll-deficient mutant of tea (Camellia sinensis).

Tea (Camellia sinensis) is a highly important beverage crop renowned for its unique flavour and health benefits. Chlorotic mutants of tea, known worldwide for their umami taste and economic value, have gained global popularity. However, the genetic basis of this chlorosis trait remains unclear. In this study, we identified a major-effect quantitative trait locus (QTL), qChl-3, responsible for the chlorosis trait in tea leaves, linked to a non-synonymous polymorphism (G1199A) in the magnesium chelatase I subunit (CsCHLI). Homozygous CsCHLIA plants exhibited an albino phenotype due to defects in magnesium protoporphyrin IX and chlorophylls in the leaves. Biochemical assays revealed that CsCHLI mutations did not affect subcellular localization or interactions with CsCHLIG and CsCHLD. However, combining CsCHLIA with CsCHLIG significantly reduced ATPase activity. RNA-seq analysis tentatively indicated that CsCHLI inhibited photosynthesis and enhanced photoinhibition, which in turn promoted protein degradation and increased the amino acid levels in chlorotic leaves. RT-qPCR and enzyme activity assays confirmed the crucial role of asparagine synthetase and arginase in asparagine and arginine accumulation, with levels increasing over 90-fold in chlorotic leaves. Therefore, this study provides insights into the genetic mechanism underlying tea chlorosis and the relationship between chlorophyll biosynthesis and amino acid metabolism.

A light-driven enzymatic enantioselective radical acylation.

Enzymes are recognized as exceptional catalysts for achieving high stereoselectivities1-3, but their ability to control the reactivity and stereoinduction of free radicals lags behind that of chemical catalysts4. Thiamine diphosphate (ThDP)-dependent enzymes5 are well-characterized systems that inspired the development of N-heterocyclic carbenes (NHCs)6-8 but have not yet been proved viable in asymmetric radical transformations. There is a lack of a biocompatible and general radical-generation mechanism, as nature prefers to avoid radicals that may be harmful to biological systems9. Here we repurpose a ThDP-dependent lyase as a stereoselective radical acyl transferase (RAT) through protein engineering and combination with organophotoredox catalysis10. Enzyme-bound ThDP-derived ketyl radicals are selectively generated through single-electron oxidation by a photoexcited organic dye and then cross-coupled with prochiral alkyl radicals with high enantioselectivity. Diverse chiral ketones are prepared from aldehydes and redox-active esters (35 examples, up to 97% enantiomeric excess (e.e.)) by this method. Mechanistic studies reveal that this previously elusive dual-enzyme catalysis/photocatalysis directs radicals with the unique ThDP cofactor and evolvable active site. This work not only expands the repertoire of biocatalysis but also provides a unique strategy for controlling radicals with enzymes, complementing existing chemical tools.

Conversion of methylmercury into inorganic mercury via organomercurial lyase (MerB) activates autophagy and aggresome formation.

Methylmercury (MeHg) is converted to inorganic mercury (iHg) in several organs; however, its impact on tissues and cells remains poorly understood. Previously, we established a bacterial organomercury lyase (MerB)-expressing mammalian cell line to overcome the low cell permeability of iHg and investigate its effects. Here, we elucidated the cytotoxic effects of the resultant iHg on autophagy and deciphered their relationship. Treatment of MerB-expressing cells with MeHg significantly increases the mRNA and protein levels of LC3B and p62, which are involved in autophagosome formation and substrate recognition, respectively. Autophagic flux assays using the autophagy inhibitor chloroquine (CQ) revealed that MeHg treatment activates autophagy in MerB-expressing cells but not in wild-type cells. Additionally, MeHg treatment induces the accumulation of ubiquitinated proteins and p62, specifically in MerB-expressing cells. Confocal microscopy revealed that large ubiquitinated protein aggregates (aggresomes) associated with p62 are formed transiently in the perinuclear region of MerB-expressing cells upon MeHg exposure. Meanwhile, inhibition of autophagic flux decreases the MeHg-induced cell viability of MerB-expressing cells. Overall, our results imply that cells regulate aggresome formation and autophagy activation by activating LC3B and p62 to prevent cytotoxicity caused by iHg. These findings provide insights into the role of autophagy against iHg-mediated toxicity.

Deciphering the Catalytic Mechanism of Virginiamycin B Lyase with Multiscale Methods and Molecular Dynamics Simulations.

Due to the emergence of antibiotic resistance, the need to explore novel antibiotics and/or novel strategies to counter antibiotic resistance is of utmost importance. In this work, we explored the molecular and mechanistic details of the degradation of a streptogramin B antibiotic by virginiamycin B (Vgb) lyase of Staphylococcus aureus using classical molecular dynamics simulations and multiscale quantum mechanics/molecular mechanics methods. Our results were in line with available experimental kinetic information. Although we were able to identify a stepwise mechanism, in the wild-type enzyme, the intermediate is short-lived, showing a small barrier to decay to the product state. The impact of point mutations on the reaction was also assessed, showing not only the importance of active site residues to the reaction catalyzed by Vgb lyase but also of near positive and negative residues surrounding the active site. Using molecular dynamics simulations, we also predicted the most likely protonation state of the 3-hydroxypicolinic moiety of the antibiotic and the impact of mutants on antibiotic binding. All this information will expand our understanding of linearization reactions of cyclic antibiotics, which are crucial for the development of novel strategies that aim to tackle antibiotic resistance.

Species-differences in the in vitro biotransformation of trifluoroethene (HFO-1123).

1,1,2-Trifluoroethene (HFO-1123) is anticipated for use as a refrigerant with low global warming potential. Inhalation studies on HFO-1123 in rats indicated a low potential for toxicity (NOAELs ≥ 20,000 ppm). In contrast, single inhalation exposure of Goettingen® minipigs (≥ 500 ppm) and New Zealand white rabbits (≥ 1250 ppm) resulted in severe toxicity. It has been suggested that these pronounced species-differences in toxicity may be attributable to species-differences in biotransformation of HFO-1123 via the mercapturic acid pathway. Therefore, the overall objective of this study was to evaluate species-differences in glutathione (GSH) dependent in vitro metabolism of HFO-1123 in susceptible versus less susceptible species and humans as a basis for human risk assessment. Biotransformation of HFO-1123 to S-(1,1,2-trifluoroethyl)-L-glutathione (1123-GSH) and subsequent cysteine S-conjugate ß-lyase-mediated cleavage of the corresponding cysteine conjugate (1123-CYS) was monitored in hepatic and renal subcellular fractions of mice, rats, minipigs, rabbits, and humans. While 1123-GSH formation occurred at higher rates in rat and rabbit liver S9 compared to minipig and human S9, increased ß-lyase cleavage of 1123-CYS was observed in minipig kidney cytosol as compared to cytosolic fractions of other species. Increased ß-lyase activity in minipig cytosol was accompanied by time-dependent formation of monofluoroacetic acid (MFA), a highly toxic compound that interferes with cellular energy production via inhibition of aconitase. Consistent with the significantly lower ß-lyase activity in human cytosols, the intensity of the MFA signal in human cytosols was only a fraction of the signal obtained in minipig subcellular fractions. Even though the inconsistencies between GSH and ß-lyase-dependent metabolism do not allow to draw a firm conclusion on the overall contribution of the mercapturic acid pathway to HFO-1123 biotransformation and toxicity in vivo, the ß-lyase data suggest that humans may be less susceptible to HFO-1123 toxicity compared to minipigs.

Steroid 17α-hydroxylase/17, 20-lyase (cytochrome P450 17A1).

Cytochrome P450 (P450) 17A1 plays a key role in steroidogenesis, in that this enzyme catalyzes the 17α-hydroxylation of both pregnenolone and progesterone, followed by a lyase reaction to cleave the C-20 land C-21 carbons from each steroid. The reactions are important in the production of both glucocorticoids and androgens. The enzyme is critical in humans but is also a drug target in treatment of prostate cancer. Detailed methods are described for the heterologous expression of human P450 17A1 in bacteria, purification of the recombinant enzyme, reconstitution of the enzyme system in the presence of cytochrome b5, and chromatographic procedures for sensitive analyses of reaction products. Historic assay approaches are reviewed. Some information is also provided about outstanding questions in the research field, including catalytic mechanisms and searches for selective inhibitors.