The Substrate Specificity of Sirtuins.

Sirtuins are NAD(+)-dependent enzymes universally present in all organisms, where they play central roles in regulating numerous biological processes. Although early studies showed that sirtuins deacetylated lysines in a reaction that consumes NAD(+), more recent studies have revealed that these enzymes can remove a variety of acyl-lysine modifications. The specificities for varied acyl modifications may thus underlie the distinct roles of the different sirtuins within a given organism. This review summarizes the structure, chemistry, and substrate specificity of sirtuins with a focus on how different sirtuins recognize distinct substrates and thus carry out specific functions.

Production of 1,4-Butanediol from Succinic Acid Using Escherichia Coli Whole-Cell Catalysis.

The widespread attention towards 1,4-butanediol (BDO) as a key chemical raw material stems from its potential in producing biodegradable plastics. However, the efficiency of its biosynthesis via current bioprocesses is limited. In this study, a dual-pathway approach for 1,4-BDO production from succinic acid was developed. Specifically, a double-enzyme catalytic pathway involving carboxylic acid reductase and ethanol dehydrogenase was proposed. Optimization of the expression levels of the pathway enzymes led to a significant 318 % increase in 1,4-BDO titer. Additionally, the rate-limiting enzyme MmCAR was engineered to enhance the kcat/KM values by 50 % and increase 1,4-BDO titer by 46.7 %. To address cofactor supply limitations, an NADPH and ATP cycling system was established, resulting in a 48.9 % increase in 1,4-BDO production. Ultimately, after 48 hours, 1,4-BDO titers reached 201 mg/L and 1555 mg/L in shake flask and 5 L fermenter, respectively. This work represents a significant advancement in 1,4-BDO synthesis from succinic acid, with potential applications in the organic chemical and food industries.

Determination of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat by ultrahigh-performance liquid chromatography-tandem mass spectrometry.

RATIONALE: Succinic acid and lactic acid have been associated with diarrhea in weaned piglets. The level of succinic acid and lactic acid in serum, meat, and intestinal contents is important to elucidate the mechanism of diarrhea in weaned piglets. METHODS: A facile method was developed for the quantification of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS). The serum samples underwent protein precipitation with methanol. The meat and intestinal contents were freeze-dried and homogenized using a tissue grinding apparatus. Methanol-water mixture (80:20, v/v) was used for homogenizing the meat, while water was used for homogenizing the intestinal contents. An additional step of protein precipitation with acetonitrile was required for the intestinal contents. The resulting solution was diluted with water before being analyzed by UHPLC/MS/MS. Separation of succinic acid and lactic acid could be achieved within 3 min using a Kinetic XB-C18 column. RESULTS: The coefficients of variation for peak areas of succinic acid and lactic acid were less than 5.0%. The established method demonstrated good linearity as indicated by correlation coefficients exceeding 0.996. Additionally, satisfactory recoveries ranging from 88.58% to 108.8% were obtained. The detection limits (RS/N = 3) for succinic acid and lactic acid were determined to be 0.75 ng/mL and 0.02 µg/mL, respectively. CONCLUSION: This method exhibited high sensitivity, simplicity in operation, and small sample weight, making it suitable for quantitative determination of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat. The method developed will provide valuable technical support in studying the metabolic mechanisms of succinic acid and lactic acid in pigs.

Elucidating the Infrared Spectral Properties of Succinic Molecular Acid Crystals: Illustration of the Structure and the Hydrogen Bond Energies of the Crystal and Its Deuterated Analogs.

Herein, the infrared spectroscopic properties of molecular succinic acid crystals (SA) and their four isotopic analogs [C2H4(COOH)2, h6-SA; C2H4(COOD)2, d2-SA; C2D4(COOH)2, d4-SA; C2D4(COOD)2, d6-SA] are reported. The correlation between the structure of succinic acid molecules and their corresponding hydrogen bond energies is elucidated. The effects related to the isotopic dilution as well as the changes in the spectrum recording temperature on the fine structures of the vO-H and vO-D bands are interpreted. The infrared spectral anomalies detected in the spectra of isotopically neat succinic nanocrystal acids are confirmed by theoretical calculations using density functional theory (DFT). According to previous spectroscopic studies of succinic acid and those carried out for α,ω-dicarboxylic acids, a decent agreement between the experimental results and the theoretical DFT simulations is obtained. Moreover, the spectra of single crystals of the h6 and d4 succinic acid variants prove that the vibrational coupling mechanism between the (COOH)2 cycles is rigorously convergent to that detected in the spectra of aromatic carboxylic acids, suggesting thereby that the promotion of symmetry-forbidden high stretching IR transitions plays a crucial role. Furthermore, the obtained experimental results reveal that the succinic acid shows a spectral behavior significantly different from that characteristic of hydrogen associations of other acids of homologous series, such as the glutaric, adipic, malonic, and pimelic acid crystals. The results obtained herein shed light on the way to explore the revealed structure of isotopic derivatives of succinic acid crystals and may prove to be useful results for understanding the nature of unconventional interactions as well as the macroscopic energy effects directing the development of hydrogen associations.

Novel chromatographic purification of succinic acid from whey fermentation broth by anionic exchange resins.

Replacement of the petroleum-based refineries with the biorefinery is regarded as an essential step towards a "zero" waste (circular) economy. Biobased succinic acid (SA) is listed by the United States Department of Energy among the top ten chemicals with the potential to replace chemicals from petroleum synthesis with renewable sources. Purification of bio-based succinic acid from fermentation by-products such as alcohols, formic acid, acetic acid and lactic is a major drawback of fermentative SA production. This study addresses this issue through a novel chromatographic separation using three distinct anionic resins: Amberlite IRA958 Cl (strong base anion exchange resin), Amberlite HPR 900 OH (strong base anion exchange resin) and Amberlyst A21 (week base anion exchange resin). The influence of process variables such as flow rate (0.18 BV/h, 0.42 BV/h and 0.84 BV/h), eluent concentration (1%, 5% and 10% HCl) and temperature (20, 30 and 40 °C) were investigated. The results indicated SA separation efficiency of 76.1%, 69.3% and 81.2% for Amberlyst A21, Amberlite HPR 900 OH and Amberlite IRA958 Cl, respectively. As the regenerant HCl concentration increased from 1 to 10%, calculated succinic acid separation efficiencies decreased from 80.3 to 70.7%. Notably, as the regenerant strength increased from 1 to 10%, the total amount of organic acids desorbed from the resin sharply increased. At operation temperatures of 20, 30 and 40 °C, SA separation efficacies were 81.2%, 73.9% and 76.4%, respectively. The insights from this study will be of great value in design of chromatographic separation systems for organic acids.

Strain-Induced Nucleophilic Ring Opening of Donor-Acceptor Cyclopropenes for Synthesis of Monosubstituted Succinic Acid Derivatives.

1,2,3-Trisubstituted donor-acceptor cyclopropenes (DACPs) generated in situ from enoldiazo compounds react with nucleophiles to form α-substituted succinic acid derivatives in high yields. Initial dirhodium(II) carboxylate catalysis rapidly converts enoldiazo-acetates or -acetamides to DACPs that undergo catalyst-free Favorskii ring opening with amines, and also with anilines, alcohols, and thiols, when facilitated by catalytic amounts of 4-dimethylaminopyridine (DMAP). This methodology provides easy access to mixed esters and amides of monosubstituted succinic acids, including derivatives of naturally occurring compounds. It also affords dihydrazide, dihydroxamic acid, and diamide derivatives, as well as α-substituted tetrahydropyridazine-3,6-diones in high yields. Attempts to generate optically enriched DACPs were not successful because their populations having the R and S configurations formed with a chiral dirhodium catalyst are quite similar, and the loss of enantiocontrol likely originates from the DACP ring forming step which is reversible with its intermediate metal carbene.

Drug-Polymer Interactions in Acetaminophen/Hydroxypropylmethylcellulose Acetyl Succinate Amorphous Solid Dispersions Revealed by Multidimensional Multinuclear Solid-State NMR Spectroscopy.

The bioavailability of insoluble crystalline active pharmaceutical ingredients (APIs) can be enhanced by formulation as amorphous solid dispersions (ASDs). One of the key factors of ASD stabilization is the formation of drug-polymer interactions at the molecular level. Here, we used a range of multidimensional and multinuclear nuclear magnetic resonance (NMR) experiments to identify these interactions in amorphous acetaminophen (paracetamol)/hydroxypropylmethylcellulose acetyl succinate (HPMC-AS) ASDs at various drug loadings. At low drug loading (<20 wt %), we showed that 1H-13C through-space heteronuclear correlation experiments identify proximity between aromatic protons in acetaminophen with cellulose backbone protons in HPMC-AS. We also show that 14N-1H heteronuclear multiple quantum coherence (HMQC) experiments are a powerful approach in probing spatial interactions in amorphous materials and establish the presence of hydrogen bonds (H-bond) between the amide nitrogen of acetaminophen with the cellulose ring methyl protons in these ASDs. In contrast, at higher drug loading (40 wt %), no acetaminophen/HPMC-AS spatial proximity was identified and domains of recrystallization of amorphous acetaminophen into its crystalline form I, the most thermodynamically stable polymorph, and form II are identified. These results provide atomic scale understanding of the interactions in the acetaminophen/HPMC-AS ASD occurring via H-bond interactions.

Understanding the binding mechanism of succinic acid against phospholipase A2 from bee venom.

Phospholipase A2 (PLA2 ) is responsible for the release of fatty acids from glycerophospholipids. PLA2 is commonly found in mammalian tissues. It is also found in venom from different animals ranging from insects, arachnid, and snakes. The release of arachidonic acid in large amount results in inflammation and pain. Identification of compounds that can inhibit the activity of PLA2 is of large scientific and medicinal interest as these compounds can act as antidotes toward snake bites and bee stings. Among the different compounds that have been tested for inhibition of PLA2 , a secondary metabolite succinic acid is identified to inhibit PLA2 activity. The inhibition was analyzed using an in vitro PLA2 inhibition assay and isothermal titration calorimetry (ITC) studies. The molecular mechanism of the mode of inhibition was studied using molecular docking and simulation studies.

Conjugate of pyrimidine derivative, the drug xymedon with succinic acid protects liver cells.

The aim of the study was to investigate the hepatoprotective properties of the conjugate of the xymedon drug substance with succinic acid (1a). The study presents an in vitro comparative evaluation of the cytotoxicity and cytoprotective properties of 1a and succinic acid on a cell line of normal human hepatocytes Chang Liver, and in vivo investigation of the ability of 1a to restore liver from the toxic damage caused by CCl4 in Wistar rats. It was shown that the cytotoxicity of 1a was 19.9 ± 0.8 mmol/L, and that of succinic acid was 14.1 ± 0.2 mmol/L. Against the background of d-galactosamine exposure, the cytoprotective effect of 1a was found to be superior to that of succinic acid. It was shown that 1a caused a significant reduction in necrotic and steatosis changes in the liver and restoration of biochemical markers of cytolysis, as well as bilirubin metabolism and synthetic liver function.

Identification of the YEATS domain of GAS41 as a pH-dependent reader of histone succinylation.

Lysine succinylation is a newly discovered posttranslational modification with distinctive physical properties. However, to date rarely have studies reported effectors capable of interpreting this modification on histones. Following our previous study of SIRT5 as an eraser of succinyl-lysine (Ksuc), here we identified the GAS41 YEATS domain as a reader of Ksuc on histones. Biochemical studies showed that the GAS41 YEATS domain presents significant binding affinity toward H3K122suc upon a protonated histidine residue. Furthermore, cellular studies showed that GAS41 had prominent interaction with H3K122suc on histones and also demonstrated the coenrichment of GAS41 and H3K122suc on the p21 promoter. To investigate the binding mechanism, we solved the crystal structure of the YEATS domain of Yaf9, the GAS41 homolog, in complex with an H3K122suc peptide that demonstrated the presence of a salt bridge formed when a protonated histidine residue (His39) recognizes the carboxyl terminal of the succinyl group. We also solved the apo structure of GAS41 YEATS domain, in which the conserved His43 residue superimposes well with His39 in the Yaf9 structure. Our findings identified a reader of succinyl-lysine, and the binding mechanism will provide insight into the development of specific regulators targeting GAS41.

Environmentally Friendly Polymer Compositions with Natural Amber Acid.

Few scientific reports have suggested the possibility of using natural phenolic acids as functional substances, such as stabilizers for polymeric materials. The replacement of commercial stabilizers in the polymer industry can be beneficial to human health and the environment. The aim of this study was to obtain biodegradable composition of polylactide (PLA) and polyhydroxyalkanoate (PHA) with natural amber (succinic) acid. The materials were subjected to controlled thermooxidation and solar aging. The research methodology included thermal analysis, examination of surface energy, mechanical properties and spectrophotometric analysis of the color change after aging. The samples of aliphatic polyesters containing from 1 to 2 parts by weight of succinic acid were characterized by increased resistance to oxidation (DSC analysis). Natural acid, preferably at a concentration of 1-1.5 parts by weight, acted as a stabilizer in the polymer compositions. On the other hand, materials that had amber acid above 2 parts by weight added were more susceptible to oxidation (DSC). They also showed the lowest aging coefficients (K). The addition of acid at 2.5-4 parts by weight caused a pro-oxidative effect and accelerated aging. By adding amber acid to PLA and PHA, it is possible to design their time in service and their overall lifetime.

Biochemical properties and crystal structure of isocitrate lyase from Bacillus cereus ATCC 14579.

The glyoxylate cycle is an important anabolic pathway and acts under a C2 compound (such as acetic acid) rich condition in bacteria. The isocitrate lyase (ICL) enzyme catalyzes the first step in the glyoxylate cycle, which is the cleavage of isocitrate to glyoxylate and succinate. This enzyme is a metalo-enzyme that contains an Mg2+ or a Mn2+ion at the active site for enzyme catalysis. We expressed and purified ICL from Bacillus cereus (BcICL) and investigated its biochemical properties and metal usage through its enzyme activity and stability with various divalent metal ion. Based on the results, BcICL mainly utilized the Mg2+ ion for enzyme catalysis as well as the Mn2+, Ni2+ and Co2+ ions. To elucidate its molecular mechanisms, we determined the crystal structure of BcICL at 1.79 Å. Through this structure, we analyzed a tetrameric interaction of the protein. We also determined the BcICL structure in complex with both the metal and its products, glyoxylate and succinate at 2.50 Å resolution and revealed each ligand binding modes.

Reducing Agent-Mediated Nonenzymatic Conversion of 2-Oxoglutarate to Succinate: Implications for Oxygenase Assays.

l-Ascorbate (l-Asc) is often added to assays with isolated FeII - and 2-oxoglutarate (2OG)-dependent oxygenases to enhance activity. l-Asc is proposed to be important in catalysis by some 2OG oxygenases in vivo. We report observations on the nonenzymatic conversion of 2OG to succinate, which is mediated by hydrogen peroxide generated by the reaction of l-Asc and dioxygen. Slow nonenzymatic oxidation of 2OG to succinate occurs with some, but not all, other reducing agents commonly used in 2OG oxygenase assays. We intend these observations will help in the robust assignment of substrates and inhibitors for 2OG oxygenases.

Cancer Cell Metabolites: Updates on Current Tracing Methods.

Cancer is the second leading cause of death-1 in 6 deaths globally is due to cancer. Cancer metabolism is a complex and one of the most actively researched area in cancer biology. Metabolic reprogramming in cancer cells entails activities that involve several enzymes and metabolites to convert nutrient into building blocks that alter energy metabolism to fuel rapid cell division. Metabolic dependencies in cancer generate signature metabolites that have key regulatory roles in tumorigenesis. In this minireview, we highlight recent advances in the popular methods ingrained in biochemistry research such as stable and flux isotope analysis, as well as radioisotope labeling, which are valuable in elucidating cancer metabolites. These methods together with analytical tools such as chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry have helped to bring about exploratory work in understanding the role of important as well as obscure metabolites in cancer cells. Information obtained from these analyses significantly contribute in the diagnosis and prognosis of tumors leading to potential therapeutic targets for cancer therapy.

Near UV and Visible Light Induce Iron-Dependent Photodegradation Reactions in Pharmaceutical Buffers: Mechanistic and Product Studies.

Near UV (λ = 320-400 nm) and visible light (λ = 400-800 nm) can lead to the oxidation of pharmaceutical proteins, which can affect efficiency and promote immunogenicity. However, no concise mechanism has been established for the photo-oxidation of pharmaceutical proteins under near UV and visible light. Here, we show that carboxylic acid buffer-Fe3+ complexes can function as photosensitizers, causing peptide degradation via the formation of various radicals and oxidants. Three pharmaceutical relevant carboxylic acid buffers (citrate, acetate, and succinate) were tested under near UV and visible light. Oxidation reactions were monitored for model peptides containing readily oxidizable amino acids, such as methionine- or leucine-enkephalin and proctolin peptide. Oxidation products were evaluated by RP-HPLC coupled to UV or fluorescent detection and RP-HPLC-MS/MS. Specifically for citrate buffer, the light-induced formation of H2O2, •OH, •CO2-, and formaldehyde was demonstrated. The peptides displayed oxidation of Met, hydroxylation of Tyr and Phe, as well as the formation of novel products from Tyr. Experiments with 18O2 resulted in the incorporation of 18O into various reaction products, consistent with a metal-catalyzed activation of O2 into reactive oxygen species. The addition of EDTA and DTPA did not prevent the oxidation of the peptides and, in some cases, enhanced the oxidation. Our results demonstrate that pharmaceutical buffer-Fe3+ complexes, exposed to UV and visible light, can promote various pathways of oxidation reactions in pharmaceutical formulations.

Synthesis and controlled drug delivery studies of a novel Ubiquinol-Polyethylene glycol-Vitamin E adduct.

CoQ10 and Vitamin E are used in medicinal applications, but they are both lipophilic molecules and the poor solubility in aqueous media results in an inefficient administration, poor bioavailability and potential toxicity. A mixed conjugate Ubiquinol-Polyethylene glycol-Vitamin E was synthesized and characterized to improve the bioavailability of CoQ10 and Vitamin E. The synthesized mixed PEG conjugate was characterized by 1H NMR spectroscopy and MALDI spectrometry. The in vitro release of the conjugate was measured at various pH conditions and in human plasma and the evaluation of free CoQ10 and Vitamin E were also conducted. The obtained results demonstrated that more CoQ10 and Vitamin E were released from PEG conjugate at pH 7.4 and in plasma within the 24 h. The antioxidant activity evaluation was carried out by DPPH assay. Our results indicated that the chemical modification after esterification with PEG of the two drugs Ubiquinol and Vitamin E doesn't significantly affected their antioxidant potential.

Structural insights into the molecular mechanism underlying Sirt5-catalyzed desuccinylation of histone peptides.

Histone modification is a ubiquitous regulatory mechanism involved in a variety of biological processes, including gene expression, DNA damage repair, cell differentiation, and ontogenesis. Succinylation sites on histones have been identified and may have functional consequences. Here, we demonstrate that human sirtuin 5 (Sirt5) catalyzes the sequence-selective desuccinylation of numerous histone succinyl sites. Structural studies of Sirt5 in complex with four succinyl peptides indicate an essential role for the conserved main chain hydrogen bonds formed by the succinyl lysine (0), +1, and +3 sites for substrate-enzyme recognition. Furthermore, biochemical assays reveal that the proline residue at the +1 site of the histone succinylation substrate is unfavorable for Sirt5 interaction. Our findings illustrate the molecular mechanism underlying the sequence-selective desuccinylase activity of Sirt5 and provide insights for further studies of the biological functions associated with histone succinylation and Sirt5.

Mechanism-based inactivator of isocitrate lyases 1 and 2 from Mycobacterium tuberculosis.

Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway for Mycobacterium tuberculosis (Mtb) during the persistent phase of human TB infection. Here, we report 2-vinyl-d-isocitrate (2-VIC) as a mechanism-based inactivator of Mtb ICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys191 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalent S-homopyruvoyl adduct of the active-site Cys191.

Inhibition of α-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors.

Two Krebs cycle genes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), are mutated in a subset of human cancers, leading to accumulation of their substrates, fumarate and succinate, respectively. Here we demonstrate that fumarate and succinate are competitive inhibitors of multiple α-ketoglutarate (α-KG)-dependent dioxygenases, including histone demethylases, prolyl hydroxylases, collagen prolyl-4-hydroxylases, and the TET (ten-eleven translocation) family of 5-methlycytosine (5mC) hydroxylases. Knockdown of FH and SDH results in elevated intracellular levels of fumarate and succinate, respectively, which act as competitors of α-KG to broadly inhibit the activity of α-KG-dependent dioxygenases. In addition, ectopic expression of tumor-derived FH and SDH mutants inhibits histone demethylation and hydroxylation of 5mC. Our study suggests that tumor-derived FH and SDH mutations accumulate fumarate and succinate, leading to enzymatic inhibition of multiple α-KG-dependent dioxygenases and consequent alterations of genome-wide histone and DNA methylation. These epigenetic alterations associated with mutations of FH and SDH likely contribute to tumorigenesis.

Nanogels of a Succinylated Glycol Chitosan-Succinyl Prednisolone Conjugate: Release Behavior, Gastrointestinal Distribution, and Systemic Absorption.

Recently, the potential of nanoparticles (NPs) in ulcerative colitis (UC) therapy has been increasingly demonstrated. Namely, anionic NPs have been found to be accumulated efficiently to the UC damaged area due to epithelial enhanced permeability and retention (eEPR) effect. Previously, a novel anionic nanogel system (NG(S)) was prepared, and evaluated for the efficacy and toxicity. In the present study, release behaviors and biodistribution were investigated in detail to elucidate the functional mechanisms. Rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced ulcerative colitis (UC) were used as biomodels. In vitro release was examined with or without the contents of the cecum or distal colon. Gastrointestinal distribution and plasma concentrations were investigated after the intragastric administration of 10 mg prednisolone (PD) eq./kg. At pH 1.2 and 6.8, release behaviors were slow, but controlled. Overall release was not markedly different irrespective of coexistence of intestinal contents. In in vivo studies, a large amount of PD was distributed in the lower parts of the gastrointestinal tract 6 and 12 h after administration with NG(S). PD accumulated well in the colonic parts, and prolonged release was noted. The systemic absorption of PD with NG(S) was hardly found. NG(S) concentrated the drug in the colon and showed controlled release. These behaviors were considered to lead to the previously reported good results, promotion of effectiveness and suppression of toxic side effects.