Structural and biochemical characterizations of Thermus thermophilus HB8 transketolase producing a heptulose.

Transketolase is a key enzyme in the pentose phosphate pathway in all organisms, recognizing sugar phosphates as substrates. Transketolase with a cofactor of thiamine pyrophosphate catalyzes the transfer of a 2-carbon unit from D-xylulose-5-phosphate to D-ribose-5-phosphate (5-carbon aldose), giving D-sedoheptulose-7-phosphate (7-carbon ketose). Transketolases can also recognize non-phosphorylated monosaccharides as substrates, and catalyze the formation of non-phosphorylated 7-carbon ketose (heptulose), which has attracted pharmaceutical attention as an inhibitor of sugar metabolism. Here, we report the structural and biochemical characterizations of transketolase from Thermus thermophilus HB8 (TtTK), a well-characterized thermophilic Gram-negative bacterium. TtTK showed marked thermostability with maximum enzyme activity at 85 °C, and efficiently catalyzed the formation of heptuloses from lithium hydroxypyruvate and four aldopentoses: D-ribose, L-lyxose, L-arabinose, and D-xylose. The X-ray structure showed that TtTK tightly forms a homodimer with more interactions between subunits compared with transketolase from other organisms, contributing to its thermal stability. A modeling study based on X-ray structures suggested that D-ribose and L-lyxose could bind to the catalytic site of TtTK to form favorable hydrogen bonds with the enzyme, explaining the high conversion rates of 41% (D-ribose) and 43% (L-lyxose) to heptulose. These results demonstrate the potential of TtTK as an enzyme producing a rare sugar of heptulose. KEY POINTS: • Transketolase catalyzes the formation of a 7-carbon sugar phosphate • Structural and biochemical characterizations of thermophilic transketolase were done • The enzyme could produce non-phosphorylated 7-carbon ketoses from sugars.

An artificial multienzyme cascade for the whole-cell synthesis of rare ketoses from glycerol.

PURPOSE: In our previous study, we constructed a one-pot multi-enzyme system for rare ketoses synthesis based on L-rhamnulose-1-phosphate aldolase (RhaD) from accessible glycerol in vitro. To eliminate tedious purification of enzymes, a facile Escherichia coli whole-cell cascade platform was established in this study. METHODS: To enhance the conversion rate, the reaction conditions, substrate concentrations and expressions of related enzymes were extensively optimized. RESULTS: The biosynthetic route for the cascade synthesis of rare ketoses in whole cells was successfully constructed and three rare ketoses including D-allulose, D-sorbose and L-fructose were produced using glycerol and D/L-glyceraldehyde (GA). Under optimized conditions, the conversion rates of rare ketoses were 85.0% and 93.0% using D-GA and L-GA as the receptor, respectively. Furthermore, alditol oxidase (AldO) was introduced to the whole-cell system to generate D-GA from glycerol, and the total production yield of D-sorbose and D-allulose was 8.2 g l-1 only from the sole carbon source glycerol. CONCLUSION: This study demonstrates a feasible and cost-efficient method for rare sugars synthesis and can also be applied to the green synthesis of other value-added chemicals from glycerol.

Enzymatic C4-Epimerization of UDP-Glucuronic Acid: Precisely Steered Rotation of a Transient 4-Keto Intermediate for an Inverted Reaction without Decarboxylation.

UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4-oxidation of the substrate by NAD+ and rotation of a decarboxylation-prone ß-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted 4 C1 chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions.

Antibacterial Target DXP Synthase Catalyzes the Cleavage of d-Xylulose 5-Phosphate: a Study of Ketose Phosphate Binding and Ketol Transfer Reaction.

The bacterial enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-dependent manner. In addition to its role in isoprenoid biosynthesis, DXP is required for ThDP and pyridoxal phosphate biosynthesis. Due to its function as a branch-point enzyme and its demonstrated substrate and catalytic promiscuity, we hypothesize that DXPS could be key for bacterial adaptation in the dynamic metabolic landscape during infection. Prior work in the Freel Meyers laboratory has illustrated that DXPS displays relaxed specificity toward donor and acceptor substrates and varies acceptor specificity according to the donor used. We have reported that DXPS forms dihydroxyethyl (DHE)ThDP from ketoacid or aldehyde donor substrates via decarboxylation and deprotonation, respectively. Here, we tested other DHE donors and found that DXPS cleaves d-xylulose 5-phosphate (X5P) at C2-C3, producing DHEThDP through a third mechanism involving d-GAP elimination. We interrogated DXPS-catalyzed reactions using X5P as a donor substrate and illustrated (1) production of a semi-stable enzyme-bound intermediate and (2) O2, H+, and d-erythrose 4-phosphate act as acceptor substrates, highlighting a new transketolase-like activity of DXPS. Furthermore, we examined X5P binding to DXPS and suggest that the d-GAP binding pocket plays a crucial role in X5P binding and turnover. Overall, this study reveals a ketose-cleavage reaction catalyzed by DXPS, highlighting the remarkable flexibility for donor substrate usage by DXPS compared to other C-C bond-forming enzymes.

Is diabetic striatopathy the culprit of seizures in a patient with ketotic hyperglycemia-induced hemichorea-hemiballismus?

BACKGROUND: Diabetic striatopathy is a rare neurological manifestation of nonketotic hyperglycemia that presents with contralateral hemichorea-hemiballismus. Presentation with concurrent seizures is rarely reported. CLINICAL PRESENTATION: We report a case of diabetic striatopathy presenting with focal and generalized tonic-clonic seizures (GTCS) with right hemichorea-hemiballismus induced by a ketotic hyperglycemic state. Head MRI showed high T1-weighted signal intensity in the left lentiform nucleus with no significant diffusion restriction or postcontrast enhancement. The patient's condition gradually improved, with seizure control on AEDs. Hemichorea-hemiballismus significantly improved with adequate blood sugar control and resolved with low-dose haloperidol. CONCLUSIONS: Diabetic striatopathy presenting with hemichorea-hemiballismus and concurrent GTCS has been reported previously in two cases; however, it has never been reported in ketotic hyperglycemia. To the best of our knowledge, we herein report the first case report of focal and generalized seizures in a ketotic hyperglycemic state and mesial temporal sclerosis.

ß-Hydroxybutyrate impairs neutrophil migration distance through activation of a protein kinase C and myosin light chain 2 signaling pathway in ketotic cows.

Ketosis in dairy cows often occurs in the peripartal period and is accompanied by immune dysfunction. High concentrations of ß-hydroxybutyrate (BHB) in peripheral blood during ketosis are closely related to the impairment of polymorphonuclear neutrophil (PMN) chemotaxis and contribute to immune dysfunction. The specific effect of BHB on PMN chemotaxis in dairy cows and the underlying molecular mechanisms are unclear. Here, 30 multiparous cows (within 3 wk postpartum) classified based on serum BHB as control (n = 15, BHB <0.6 mM) or clinically ketotic (n = 15, BHB >3.0 mM) were used. Blood samples were collected before feeding, and the isolated PMN were treated with platelet-activating factor for 0.5 h to activate their migration. Scanning electron microscopy revealed a longer tail in PMN of ketotic cows. In addition, the phosphorylation and transcription levels of myosin light chain 2 (MLC2) increased in PMN of ketotic cows. Polymorphonuclear neutrophils from control dairy cows were incubated with 3.0 mM BHB for different times in vitro, and 6 h was selected as the proper duration of BHB stimulation according to its inhibition effect on PMN migration using an under-agarose PMN chemotaxis model. Similarly, BHB stimulation in vitro resulted in inhibition of migration distance and deviation of migration direction of PMN, as well as a longer tail in morphology in the scanning electron microscope data, suggesting that BHB-induced PMN migration inhibition may be mediated by impairing the trailing edge contraction. To confirm this hypothesis, sotrastaurin (Sotra)-a specific inhibitor of protein kinase C (PKC), which is the core regulator of cell contraction-was used with or without BHB treatment in vitro. Sotra was pretreated 0.5 h before BHB treatment. Accordingly, BHB treatment increased the phosphorylation level of PKC and MLC2, the protein abundance of RhoA and rho-kinase 1 (ROCK1), and the mRNA abundance of PRKCA, MYL2, RHOA, and ROCK1 in PMN. In contrast, these effects of BHB on PMN were dampened by Sotra. As demonstrated by immunofluorescence experiments in vitro, the BHB-induced inhibition of trailing edge contraction of PMN was relieved by Sotra. In addition, Sotra also dampened the effects of BHB on PMN migration in vitro. Furthermore, as verified by in vivo experiments, compared with the control cows, both abundance and activation of PKC signaling were enhanced in PMN of ketotic cows. Overall, the present study revealed that high concentrations of blood BHB impaired PMN migration distance through inhibition of the trailing edge contraction, mediated by enhancing the activation of PKC-MLC2 signaling. These findings help explain the dysfunctional immune state in ketotic cows and provide information on the pathogenesis of infectious diseases secondary to ketosis.

Effects of diacylglycerol O-acyltransferase 1 (DGAT1) on endoplasmic reticulum stress and inflammatory responses in adipose tissue of ketotic dairy cows.

Adipose tissue of ketotic dairy cows exhibits greater lipolytic rate and signs of inflammation, which further aggravate the metabolic disorder. In nonruminants, the endoplasmic reticulum (ER) is a key organelle coordinating metabolic adaptations and cellular functions; thus, disturbances known as ER stress lead to inflammation and contribute to metabolic disorders. Enhanced activity of diacylglycerol O-acyltransferase 1 (DGAT1) in murine adipocytes undergoing lipolysis alleviated ER stress and inflammation. The aim of the present study was to investigate the potential role of DGAT1 on ER stress and inflammatory response of bovine adipose tissue in vivo and in vitro. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of ß-hydroxybutyrate, which were 4.05 (interquartile range = 0.46) and 0.52 mM (interquartile range = 0.14), respectively. Protein abundance of DGAT1 was greater in adipose tissue of ketotic cows. Among ER stress proteins measured, ratios of phosphorylated PKR-like ER kinase (p-PERK) to PERK and phosphorylated inositol-requiring enzyme 1 (p-IRE1) to IRE1, and protein abundance of cleaved ATF6 protein were greater in adipose tissue of ketotic cows. Furthermore, ratios of phosphorylated RELA subunit of NF-κB (p-RELA) to RELA and phosphorylated c-jun N-terminal kinase (p-JNK) to JNK were greater, whereas protein abundance of NF-κB inhibitor α (NFKBIA) was lower in adipose tissue of ketotic cows. In addition, mRNA abundance of proinflammatory cytokines including TNF and IL-6 was greater in adipose tissue of ketotic cows. To better address mechanistic aspects of these responses, primary bovine adipocytes isolated from the harvested adipose tissue of healthy cows were subjected to lipolysis-stimulating conditions via incubation with 1 µM epinephrine (EPI) for 2 h. In another experiment, adipocytes were cultured with DGAT1 overexpression adenovirus and DGAT1 small interfering RNA for 48 h, respectively, followed by EPI (1 µM) exposure for 2 h. Treatment with EPI led to greater ratios of p-PERK to PERK, p-IRE1 to IRE1, p-RELA to RELA, p-JNK to JNK, and cleaved ATF6 protein, whereas EPI stimulation inhibited protein abundance of NFKBIA. Furthermore, treatment with EPI upregulated the secretion of proinflammatory cytokines into culture medium, including TNF-α and IL-6. Overexpression of DGAT1 in EPI-treated adipocytes attenuated ER stress, the activation of NF-κB and JNK signaling pathways, and the secretion of inflammatory cytokines. In contrast, silencing DGAT1 further aggravated EPI-induced ER stress and inflammatory responses. Overall, these data indicated that activation of DGAT1 may act as an adaptive mechanism to dampen metabolic dysregulation in adipose tissue. As such, it contributes to relief from ER stress and inflammatory responses.

Disruption of endoplasmic reticulum homeostasis exacerbates liver injury in clinically ketotic cows.

Disruption of endoplasmic reticulum (ER) homeostasis, a condition termed "ER stress," contributes to the development of liver injury in nonruminants. Because liver injury is a prominent pathological feature associated with overproduction of ketone bodies in dairy cows with ketosis, understanding the ER stress state and its functional consequences on liver injury is of particular interest. Here, 30 multiparous cows (within 3 wk postpartum) classified based on blood ß-hydroxybutyrate (BHB) as healthy (n = 15, BHB <0.6 mM) or clinically ketotic (n = 15, BHB >3.0 mM) were used. Compared with healthy cows, ketotic cows had greater levels of serum fatty acids and activities of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyl transferase, and glutamate dehydrogenase but lower serum glucose. Furthermore, dairy cows with ketosis had greater protein abundance of ER stress markers in liver tissue, including protein kinase RNA-like ER kinase (PERK), inositol-requiring protein-1α (IRE1α), and cleaved activating transcription factor-6 (ATF6). Cows with ketosis also had higher mRNA levels of hepatic 78-kDa glucose-regulated protein (GRP78) and spliced X-box binding protein 1 (sXBP1). These data confirmed an enhanced ER stress state in clinically ketotic cows. To explore whether enhanced hepatic ER stress was induced by elevated ketone bodies and the possible contribution of ER stress to liver injury, in vitro experiments were then performed using isolated primary calf hepatocytes treated with incremental concentrations of BHB (0, 0.6, 1.2, 3.0, and 4.8 mM) for 12 h with or without overexpression of GRP78 (the master regulator of unfolded protein response). Phosphorylation levels of PERK and IRE1α proteins, level of cleaved ATF6 protein, and mRNA abundance of GRP78 and sXBP1 in hepatocytes increased after treatment with high (3.0 and 4.8 mM) BHB, indicating a mechanistic link between excessive BHB and enhanced hepatic ER stress. Furthermore, treatment with 3.0 and 4.8 mM BHB markedly elevated activities of aspartate aminotransferase and alanine aminotransferase in cell supernatant, indicating exacerbated hepatocyte damage after ER stress was enhanced. Overexpression of GRP78 attenuated both BHB-induced ER stress and the ensuing cellular damage, suggesting that hepatocyte damage caused by excessive BHB can be mediated via enhanced ER stress. Overall, the present study revealed that ER stress may exacerbate liver injury development in clinically ketotic cows, underscoring the biological relevance of this pathway in the context of liver injury.

Food-derived 1,2-dicarbonyl compounds and their role in diseases.

Reactive 1,2-dicarbonyl compounds (DCs) are generated from carbohydrates during food processing and storage and under physiological conditions. In the recent decades, much knowledge has been gained concerning the chemical formation pathways and the role of DCs in food and physiological systems. DCs are formed mainly by dehydration and redox reactions and have a strong impact on the palatability of food, because they participate in aroma and color formation. However, they are precursors of advanced glycation end products (AGEs), and cytotoxic effects of several DCs have been reported. The most abundant DCs in food are 3-deoxyglucosone, 3-deoxygalactosone, and glucosone, predominating over methylglyoxal, glyoxal, and 3,4-dideoxyglucosone-3-ene. The availability for absorption of individual DCs is influenced by the release from the food matrix during digestion and by their reactivity towards constituents of intestinal fluids. Some recent works suggest formation of DCs from dietary sugars after their absorption, and others indicate that certain food constituents may scavenge endogenously formed DCs. First works on the interplay between dietary DCs and diseases reveal an ambiguous role of the compounds. Cancer-promoting but also anticancer effects were ascribed to methylglyoxal. Further work is still needed to elucidate the reactions of DCs during intestinal digestion and pathophysiological effects of dietary DCs at doses taken up with food and in "real" food matrices in disease states such as diabetes, uremia, and cancer.

Construction of a fusion enzyme for astaxanthin formation and its characterisation in microbial and plant hosts: A new tool for engineering ketocarotenoids.

The high-value ketocarotenoid astaxanthin, a natural red colorant with powerful antioxidant activity, is synthesised from ß-carotene by a hydroxylase and an oxygenase enzyme, which perform the addition of two hydroxyl and keto moieties, respectively. Several routes of intermediates, depending on the sequence of action of these enzymes, lead to the formation of astaxanthin. In the present study, the enzyme activities of 3, 3' ß-carotene hydroxylase (CRTZ) and 4, 4' ß-carotene oxygenase (CRTW) have been combined through the creation of "new to nature" enzyme fusions in order to overcome leakage of non-endogenous intermediates and pleotropic effects associated with their high levels in plants. The utility of flexible linker sequences of varying size has been assessed in the construction of pZ-W enzyme fusions. Frist, in vivo color complementation assays in Escherichia coli have been used to evaluate the potential of the fusion enzymes. Analysis of the carotenoid pigments present in strains generated indicated that the enzyme fusions only possess both catalytic activities when CRTZ is attached as the N-terminal module. Astaxanthin levels in E. coli cells were increased by 1.4-fold when the CRTZ and CRTW enzymes were fused compared to the individual enzymes. Transient expression in Nicotiana benthamiana was then performed in order to assess the potential of the fusions in a plant system. The production of valuable ketocarotenoids was achieved using this plant-based transient expression system. This revealed that CRTZ and CRTW, transiently expressed as a fusion, accumulated similar levels of astaxanthin compared to the expression of the individual enzymes whilst being associated with reduced ketocarotenoid intermediate levels (e.g. phoenicoxanthin, canthaxanthin and 3-OH-echinenone) and a reduced rate of leaf senescence after transformation. Therefore, the quality of the plant material producing the ketocarotenoids was enhanced due to a reduction in the stress induced by the accumulation of high levels of heterologous ketocarotenoid intermediates. The size of the linkers appeared to have no effect upon activity. The potential of the approach to production of valuable plant derived products is discussed.

Direct glucosone-based synthesis and HILIC-ESI-MS/MS characterization of N-terminal fructosylated valine and valylhistidine for validation of enzymatic HbA1c assays in the diagnosis of diabetes mellitus.

Naturally occurring fructosamines are of high clinical significance due to their potential use in diabetes mellitus monitoring (quantification of fructosylated hemoglobin, HbA1c) or for the investigation of their reactivity in consecutive reactions and harmfulness towards the organism. Here we report the specific synthesis of the fructosylated dipeptide L-valyl-L-histidine (Fru-Val-His) and fructosylated L-valine (Fru-Val). Both are basic tools for the development and validation of enzymatic HbA1c assays. The two fructosamine derivatives were synthesized via a protected glucosone intermediate which was coupled to the primary amine of Val or Val-His, performing a reductive amination reaction. Overall yields starting from fructose were 36% and 34% for Fru-Val and Fru-Val-His, respectively. Both compounds were achieved in purities > 90%. A HILIC-ESI-MS/MS method was developed for routine analysis of the synthesized fructosamines, including starting materials and intermediates. The presented method provides a well-defined and efficient synthesis protocol with purification steps and characterization of the desired products. The functionality of the fructosylated dipeptide has been thoroughly tested in an enzymatic HbA1c assay, showing its concentration-dependent oxidative degradation by fructosyl-peptide oxidases (FPOX). Graphical abstract.

Study of Aberrant Modifications in Peptides as a Test Bench to Investigate the Immunological Response to Non-Enzymatic Glycation.

A side effect of diabetes is formation of glycated proteins and, from them, production of advanced early glycation end products that could determine aberrant immune responses at the systemic level. We investigated a relevant aberrant post-translational modification (PTM) in diabetes based on synthetic peptides modified on the lysine side chain residues with 1-deoxyfructopyranosyl moiety as a possible modification related to glycation. The PTM peptides were used as molecular probes for detection of possible specific autoantibodies developed by diabetic patients. The PDC-E2(167-186) sequence from the pyruvate dehydrogenase complex was selected and tested as a candidate peptide for antibody detection. The structure-based designed type I' ß-turn CSF114 peptide was also used as a synthetic scaffold. Twenty-seven consecutive type 1 diabetic patients and 29 healthy controls were recruited for the study. In principle, the 'chemical reverse approach', based on the use of patient sera to screen the synthetic modified peptides, leads to the identification of specific probes able to characterize highly specific autoantibodies as disease biomarkers of autoimmune disorders. Quite surprisingly, both peptides modified with the (1-deoxyfructosyl)-lysine did not lead to significant results. Both IgG and IgM differences between the two populations were not significant. These data can be rationalized considering that i) IgGs in diabetic subjects exhibit a high degree of glycation, leading to decreased functionality; ii) IgGs in diabetic subjects exhibit a privileged response vs proteins containing advanced glycation products (e.g., methylglyoxal, glyoxal, glucosone, hydroimidazolone, dihydroxyimidazolidine) and only a minor one with respect to (1-deoxyfructosyl)-lysine.

Extending the spectrum of α-dicarbonyl compounds in vivo.

Maillard α-dicarbonyl compounds are known as central intermediates in advanced glycation end product (AGE) formation. Glucose is the primary source of energy for the human body, whereas l-threo-ascorbic acid (vitamin C) is an essential nutrient, involved in a variety of enzymatic reactions. Thus, the Maillard degradation of glucose and ascorbic acid is of major importance in vivo. To understand the complex mechanistic pathways of AGE formation, it is crucial to extend the knowledge on plasma concentrations of reactive key α-dicarbonyl compounds (e.g. 1-deoxyglucosone). With the present work, we introduce a highly sensitive LC-MS/MS multimethod for human blood plasma based on derivatization with o-phenylenediamine under acidic conditions. The impact of workup and reaction conditions, particularly of pH, was thoroughly evaluated. A comprehensive validation provided the limit of detection, limit of quantitation, coefficients of variation, and recovery rates. The method includes the α-dicarbonyls 1-deoxyglucosone, 3-deoxyglucosone, glucosone, Lederer's glucosone, dehydroascorbic acid, 2,3-diketogulonic acid, 1-deoxypentosone, 3-deoxypentosone, 3,4-dideoxypentosone, pentosone, 1-deoxythreosone, 3-deoxythreosone, threosone, methylglyoxal, glyoxal; the α-keto-carboxylic acids pyruvic acid and glyoxylic acid; and the dicarboxylic acid oxalic acid. The method was then applied to the analyses of 15 healthy subjects and 24 uremic patients undergoing hemodialysis. The comparison of the results revealed a clear shift in the product spectrum. In most cases, the plasma levels of target analytes were significantly higher. Thus, this is the first time that a complete spectrum of α-dicarbonyl compounds relevant in vivo has been established. The results provide further insights into the chemistry of AGE formation and will be helpful to find specific markers to differentiate between the various precursors of glycation.

When Good Intentions Go Awry: Modification of a Recombinant Monoclonal Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbic Acid.

With the advent of new initiatives to develop chemically defined media, cell culture scientists screen many additives to improve cell growth and productivity. However, the introduction or increase of supplements, typically considered beneficial or protective on their own, to the basal media or feed stream may cause unexpected detrimental consequences to product quality. For instance, because cultured cells are constantly under oxidative stress, ascorbic acid (vitamin C, a potent natural reducing agent) is a common additive to cell culture media. However, as reported herein, a recombinant monoclonal antibody (adalimumab) in cell culture was covalently modified by xylosone (molecular weight 148), an oxidative product of ascorbate. Containing reactive carbonyl groups, xylosone modifies various amines (e.g., the N-termini of the heavy and light chains and susceptible lysines), forming either hemiaminal (+148 Da) or Schiff base (imine, +130 Da) products. Our findings show, for the first time, that ascorbate-derived xylosone can contribute to an increase in molecular heterogeneity, such as acidic species. Our work serves as a reminder that additives to cell culture and their metabolites may become reactive and negatively impact the overall product quality and should be carefully monitored with any changes in cell culture conditions.

Near-Infrared Fluorescence Probe for Monitoring the Metabolic Products of Vitamin C in HepG2 Cells under Normoxia and Hypoxia.

Vitamin C (ascorbic acid; AA) is a well-known reducing agent and has been evaluated for its antitumor activity. However, the mechanism for its antitumor action remains unclear. Tracking the metabolism of AA may help to elucidate its antitumor mechanism. In this study, a near-infrared fluorescent probe (Arg-Cy) for monitoring the metabolic products of AA in living cells was developed based on the reaction of the guanidine group in Arg-Cy with the adjacent diketone involved in the metabolites of AA. Consequently, the probe can respond to L-xylosone, a metabolite of AA, with high selectivity and sensitivity and was successfully used to visualize the real-time changes of L-xylosone levels in living cells incubated under normoxic conditions. Considering that the tumor microenvironment suffers from hypoxia, the L-xylosone levels in the process of HepG2 cell death induced by pharmacological doses of AA were also monitored under hypoxic conditions. Surprisingly, no obvious fluorescence change appeared during this process. Furthermore, detection of the intracellular redox state using a reported H2O2 probe confirmed that AA can be metabolized to L-xylosone only under normoxic conditions due to the oxidative stress, but not under hypoxic conditions. Therefore, we hypothesize that the mechanism for cell death induced by AA under hypoxia is different from that under normoxia. Thus, the developed probe can provide a tool for monitoring the metabolism of AA and may help to clarify the mechanism for the antitumor activity of vitamin C in the tumor microenvironment.

Biosynthesis of rare ketoses through constructing a recombination pathway in an engineered Corynebacterium glutamicum.

Rare sugars have various known biological functions and potential for applications in pharmaceutical, cosmetics, and food industries. Here we designed and constructed a recombination pathway in Corynebacterium glutamicum, in which dihydroxyacetone phosphate (DHAP), an intermediate of the glycolytic pathway, and a variety of aldehydes were condensed to synthesize rare ketoses sequentially by rhamnulose-1-phosphate aldolase (RhaD) and fructose-1-phosphatase (YqaB) obtained from Escherichia coli. A wild-type strain harboring this artificial pathway had the ability to produce D-sorbose and D-psicose using D-glyceraldehyde and glucose as the substrates. The tpi gene, encoding triose phosphate isomerase was further deleted, and the concentration of DHAP increased to nearly 20-fold relative to that of the wild-type. After additional optimization of expression levels from rhaD and yqaB genes and of the fermentation conditions, the engineered strain SY6(pVRTY) exhibited preferable performance for rare ketoses production. Its yield increased to 0.59 mol/mol D-glyceraldehyde from 0.33 mol/mol D-glyceraldehyde and productivity to 2.35 g/L h from 0.58 g/L h. Moreover, this strain accumulated 19.5 g/L of D-sorbose and 13.4 g/L of D-psicose using a fed-batch culture mode under the optimal conditions. In addition, it was verified that the strain SY6(pVRTY) meanwhile had the ability to synthesize C4, C5, C6, and C7 rare ketoses when a range of representative achiral and homochiral aldehydes were applied as the substrates. Therefore, the platform strain exhibited the potential for microbial production of rare ketoses and deoxysugars.

Multicomponent Cascade Reactions of Unprotected Ketoses and Amino Acids--Access to a Defined Configured Quaternary Stereogenic Center.

A highly stereoselective multicomponent cascade reaction of ketones with unprotected amino acids was developed. This operationally simple methodology was expanded to reactions of unprotected ketohexoses and unprotected amino acids. By the careful choice of amino acid and isonitrile, an optional access to all possible enantiomers is given.

Ultrafast time resolved spectroscopic studies on the generation of the ketyl-sugar biradical by intramolecular hydrogen abstraction among ketoprofen and purine nucleoside dyads.

Intramolecular hydrogen abstraction reactions among ketoprofen (KP) and purine nucleoside dyads have been proposed to form ketyl-sugar biradical intermediates in acetonitrile. Femtosecond transient absorption studies on KP and purine nucleoside dyads reveal that the triplet state of the KP moiety of the dyads with cisoid structure decay faster (due to an intramolecular hydrogen abstraction reaction to produce a ketyl-sugar biradical intermediate) than the triplet state of the KP moiety of the dyads with transoid structure detected in acetonitrile solvent. For the cisoid 5-KP-dG dyad, the triplet state of the KP moiety decays too fast to be observed by ns-TR(3); only the ketyl-sugar biradical intermediates are detected by ns-TR(3) in acetonitrile. For the cisoid 5-KP-dA dyad, the triplet states of the KP moiety could be observed at early nanosecond delay times, and then it quickly undergoes intramolecular hydrogen abstraction to produce a ketyl-sugar biradical intermediate. For the cisoid 5-KPGly-dA and transoid 3-KP-dA dyads, the triplet state of the KP moiety had a longer lifetime due to the long distance chain between the KP moiety and the purine nucleoside (5-KPGly-dA) and the transoid structure (3-KP-dA). The experimental and computational results suggest that the ketyl-sugar biradical intermediate is generated with a higher efficiency for the cisoid dyad. However, the transoid dyad exhibits similar photochemistry behavior as the KP molecule, and no ketyl-sugar biradical intermediate was observed in the ns-TR(3) experiments for the transoid 3-KP-dA dyad.

Characterization of glycerol phosphate oxidase from Streptococcus pneumoniae and its application for ketose synthesis.

Glycerol phosphate oxidase from Streptococcus pneumoniae (GPOS.pne) was purified and characterized. By the actions of GPOS.pne and dihydroxyacetone phosphate (DHAP)-dependent aldolases, various ketoses including rare sugars were synthesized with glyceraldehydes as acceptors in a one-pot four-enzyme system.