Pyruvate Formation Research Articles

Solution structure and biochemical characterization of a spare part protein that restores activity to an oxygen-damaged glycyl radical enzyme.

Glycyl radical enzymes (GREs) utilize a glycyl radical cofactor to carry out a diverse array of chemically challenging enzymatic reactions in anaerobic bacteria. Although the glycyl radical is a powerful catalyst, it is also oxygen sensitive such that oxygen exposure causes cleavage of the GRE at the site of the radical. This oxygen sensitivity presents a challenge to facultative anaerobes dwelling in areas prone to oxygen exposure. Once GREs are irreversibly oxygen damaged, cells either need to make new GREs or somehow repair the damaged one. One particular GRE, pyruvate formate lyase (PFL), can be repaired through the binding of a 14.3kDa protein, termed YfiD, which is constitutively expressed in E. coli. Herein, we have solved a solution structure of this 'spare part' protein using nuclear magnetic resonance spectroscopy. These data, coupled with data from circular dichroism, indicate that YfiD has an inherently flexible N-terminal region (residues 1-60) that is followed by a C-terminal region (residues 72-127) that has high similarity to the glycyl radical domain of PFL. Reconstitution of PFL activity requires that YfiD binds within the core of the PFL barrel fold; however, modeling suggests that oxygen-damaged, i.e. cleaved, PFL cannot fully accommodate YfiD. We further report that a PFL variant that mimics the oxygen-damaged enzyme is highly susceptible to proteolysis, yielding additionally truncated forms of PFL. One such PFL variant of ~ 77kDa makes an ideal scaffold for the accommodation of YfiD. A molecular model for the rescue of PFL activity by YfiD is presented.

Distribution of Yeast Cells, Temperature, and Fermentation By-Products in White Wine Fermentations

Yeast inoculation from the tops of tanks is common in white wine fermentations. Our objective was to examine whether inhomogeneities occur in tanks following yeast addition from the top with and without brief stirring after inoculation. We investigated whether inhomogeneities affect fermentation kinetics and formation of acetaldehyde, acetic acid, and pyruvate in Riesling juices with low turbidity. Samples and temperatures were taken at multiple tank heights in pilot-scale (105 L) and industrial-scale (2500 and 7000 L) tanks to investigate the distributions of temperature, yeast, and fermentation products in tanks of different volumes and height-to-diameter ratios. When the tank was not stirred following inoculation, cell counts were higher in the upper tank section for multiple days. Fermentation started earlier at the top of the tank than at lower levels, as indicated by specific gravity measurements. The top and bottom of the tanks differed by up to 8.6°C and 0.040 in specific gravity. The inhomogeneities lasted four days in the 7000 L tanks. These results suggest slow yeast sedimentation and poor mixing in the downward direction during the early fermentation phase. During vigorous fermentation, all parameters were homogenously distributed. Brief stirring after yeast inoculation provided uniform conditions during the early fermentation phase, except for temperature differences due to natural stratification. Stirring also shortened the fermentation duration. The final pyruvate concentration was significantly lower when the yeast was stirred in, presumably due to better nutrient availability throughout the tank, reducing sulfur dioxide demand. No differences were observed in acetaldehyde and acetic acid concentrations. When yeast is not stirred in following inoculation, tank sampling valves and temperature sensors do not reflect the entire tank during the early fermentation phase, which will negatively affect process control.

The Influence of Chemical Change on Protein Dynamics: A Case Study with Pyruvate Formate‐Lyase

AbstractInvited for the cover of this issue is David M. Smith and co‐workers at the Ruđer Bošković Institute, Zagreb, Croatia and Friedrich Alexander University Erlangen Nurnberg, Germany. The image depicts coenzyme A reaching for the acetyl group in the active site of pyruvate formate–lyase. Read the full text of the article at 10.1002/chem.201900663.

Effect of Respiratory Growth on the Metabolite Production and Stress Robustness of Lactobacillus casei N87 Cultivated in Cheese Whey Permeate Medium.

Cheese whey permeate (WP) is a low-cost feedstock used for the production of biomass and metabolites from several lactic acid bacteria (LAB) strains. In this study, Lactobacillus casei N87 was cultivated in an optimized WP medium (WPM) to evaluate the effect of anaerobic and respiratory conditions on the growth performances (kinetics, biomass yield), consumption of sugars (lactose, galactose, glucose) and citrate, metabolite production [organic acids, volatile organic compounds (VOCs)] and stress survival (oxidative, heat, freezing, freeze-drying). The transcription of genes involved in the main pathways for pyruvate conversion was quantified through Real Time-PCR to elucidate the metabolic shifts due to respiratory state. Cultivation in WPM induced a diauxic growth in both anaerobic and respiratory conditions, and L. casei N87 effectively consumed the lactose and galactose present in WPM. Genomic information suggested that membrane PTS system and tagatose-6-P pathway mediated the metabolism of lactose and galactose in L. casei N87. Respiration did not affect specific growth rate and biomass production, but significantly altered the pyruvate conversion pathways, reducing lactate accumulation and promoting the formation of acetate, acetoin and diacetyl to ensure the redox balance. Ethanol was not produced under either cultivation. Pyruvate oxidase (pox), acetate kinase (ack), α-acetolactate decarboxylase (ald), acetolactate synthase (als) and oxaloacetate decarboxylase (oad) genes were up-regulated under respiration, while L-lactate dehydrogenase (ldh), pyruvate formate lyase (pfl), pyruvate carboxylase (pyc), and phosphate acetyltransferase (pta) were down regulated by oxygen. Transcription analysis was consistent with metabolite production, confirming that POX-ACK and ALS-ALD were the alternative pathways activated under aerobic cultivation. Respiratory growth affected the production of volatile compounds useful for the development of aroma profile in several fermented foods, and promoted the survival of L. casei N87 to oxidative stresses and long-term storage. This study confirmed that the respiration-based technology coupled with cultivation on low-cost medium may be effectively exploited to produce competitive and functional starter and/or adjunct cultures. Our results, additionally, provided further information on the activation and regulation of metabolic pathways in homofermentative LAB grown under respiratory promoting conditions.

The Response to Stimulation in Neurons and Astrocytes.

The brain uses mainly glucose as fuel with an index of glucose to oxygen utilization close to 6, the maximal index if all glucose was completely oxidized. However, this high oxidative index, contrasts with the metabolic traits of the major cell types in the brain studied in culture, neurons and astrocytes, including the selective use of the malate-aspartate shuttle (MAS) in neurons and the glycerol-phosphate shuttle in astrocytes. Metabolic interactions among these cell types may partly explain the high oxidative index of the brain. In vivo, neuronal activation results in a decrease in the oxygen glucose index, which has been attributed to a stimulation of glycolysis and lactate production in astrocytes in response to glutamate uptake (astrocyte-neuron lactate shuttle, ANLS). Recent findings indicate that this is accompanied with a stimulation of pyruvate formation and astrocyte respiration, indicating that lactate formation is not the only astrocytic response to neuronal activation. ANLS proposes that neurons utilize lactate produced by neighboring astrocytes. Indeed, neurons can use lactate to support an increase in respiration with different workloads, and this depends on the Ca2+ activation of MAS. However, whether this activation operates in the brain, particularly at high stimulation conditions, remains to be established.

MTORC1 is required for expression of LRPPRC and cytochrome-c oxidase but not HIF-1α in Leigh syndrome French Canadian type patient fibroblasts.

Leigh syndrome French Canadian type (LSFC) is a mitochondrial disease caused by mutations in the leucine-rich pentatricopeptide repeat-containing (LRPPRC) gene leading to a reduction of cytochrome-c oxidase (COX) expression reaching 50% in skin fibroblasts. We have shown that under basal conditions, LSFC and control cells display similar ATP levels. We hypothesized that this occurs through upregulation of mechanistic target of rapamycin (mTOR)-mediated metabolic reprogramming. Our results showed that compared with controls, LSFC cells exhibited an upregulation of the mTOR complex 1 (mTORC1)/p70 ribosomal S6 kinase pathway and higher levels of hypoxia-inducible factor 1α (HIF-1α) and its downstream target pyruvate dehydrogenase kinase 1 (PDHK1), a regulator of mitochondrial pyruvate dehydrogenase 1 (PDH1). Consistent with these signaling alterations, LSFC cells displayed a 40-61% increase in [U-13C6]glucose contribution to pyruvate, lactate, and alanine formation, as well as higher levels of the phosphorylated and inactive form of PDH1-α. Interestingly, inhibition of mTOR with rapamycin did not alter HIF-1α or PDHK1 protein levels in LSFC fibroblasts. However, this treatment increased PDH1-α phosphorylation in control and LSFC cells and reduced ATP levels in control cells. Rapamycin also decreased LRPPRC expression by 41 and 11% in LSFC and control cells, respectively, and selectively reduced COX subunit IV expression in LSFC fibroblasts. Taken together, our data demonstrate the importance of mTORC1, independent of the HIF-1α/PDHK1 axis, in maintaining LRPPRC and COX expression in LSFC cells.

Metabolic engineering of the type I methanotroph Methylomonas sp. DH-1 for production of succinate from methane

Methane-utilizing methanotrophs are fascinating systems for methane bioconversion. Methylomonas sp. DH-1, a novel type I methanotroph isolated from brewery sludge, has been evaluated as a promising candidate for an industrial bio-catalyst. Succinate has been considered one of the top building block chemicals for the agricultural, food, and pharmaceutical industries. In this study, Methylomonas sp. DH-1 was engineered to accumulate succinate as a desired product. The TCA cycle and enzymes diverting carbon flux to acetate or formate were modified or deleted to improve succinate productivity. By deleting succinate dehydrogenase (sdh) in the TCA cycle, succinate production increased dramatically ∼10 times compared to that of the wild type. In addition, the maximum succinate titer of ∼134 mg/L (DS-GL) was achieved by integrating glyoxylate shunt enzymes from the E. coli MG1655 strain. Pyruvate formate lyase (pfl) and acetate kinase-phosphotransacetylase (ack-pta) genes were disrupted to further concentrate carbon flux to the TCA cycle. However, these additional disruptions of competitive pathways did not affect cell growth or succinate production positively. The mutant strain DS-GL, which showed the best succinate production, was grown in a fed-batch bioreactor, and higher cell growth and succinate production (∼195 mg/L succinate with 0.0789 g-succinate/g-methane yield) were achieved. In this study, we demonstrated a novel platform for microbial conversion of methane to succinate using methanotroph.

Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana.

Since nitrogenase is extremely vulnerable to oxygen, aerobic or micro-aerobic nitrogen-fixing organisms need to create anaerobic microenvironments in the cells for diazotrophic growth, which would be one of the major barriers to express active nitrogenase in plants in efforts to create nitrogen-fixing plants. Numerous cyanobacteria are able to fix nitrogen with nitrogenase by coping with the endogenous oxygen production by photosynthesis. Understanding of the molecular mechanisms enabling to the coexistence of nitrogen fixation and photosynthesis in nonheterocystous cyanobacteria could offer valuable insights for the transfer of nitrogen fixation capacity into plants. We previously identified the cnfR gene encoding the master regulator for the nitrogen fixation (nif) gene cluster in the genome of a nonheterocystous cyanobacterium Leptolyngbya boryana, in addition to initial characterization of the nif gene cluster. Here we isolated nine mutants, in which the nif and nif-related genes were individually knocked out in L. boryana to investigate the individual functions of (1) accessory proteins (NifW, NifX/NafY, and NifZ) in the biosynthesis of nitrogenase metallocenters, (2) serine acetyltransferase (NifP) in cysteine supply for iron-sulfur clusters, (3) pyruvate formate lyase in anaerobic metabolism, and (4) NifT and HesAB proteins. ΔnifW, ΔnifXnafY, and ΔnifZ exhibited the most severe phenotype characterized by low nitrogenase activity (<10%) and loss of diazotrophic growth ability. The phenotypes of ΔnifX, ΔnafY, and ΔnifXnafY suggested that the functions of the homologous proteins NifX and NafY partially overlap. ΔnifP exhibited significantly slower diazotrophic growth than the wild type, with lower nitrogenase activity (22%). The other four mutants (ΔpflB, ΔnifT, ΔhesA, and ΔhesB) grew diazotrophically similar to the wild type. Western blot analysis revealed a high correlation between nitrogenase activity and NifD contents, suggesting that NifD is more susceptible to proteolytic degradation than NifK in L. boryana. The phenotype of the mutants lacking the accessory proteins was more severe than that observed in heterotrophic bacteria such as Azotobacter vinelandii, which suggests that the functions of NifW, NifX/NafY, and NifZ are critical for diazotrophic growth of oxygenic photosynthetic cells. L. boryana provides a promising model for studying the molecular mechanisms that produce active nitrogenase, to facilitate the creation of nitrogen-fixing plants.

Iron Regulation in Clostridioides difficile

The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen Clostridioides difficile were investigated by a combined RNA sequencing, proteomic, metabolomic and electron microscopy approach. At high iron conditions (15 μM) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron and siderophore transporter genes were induced by Fur during low iron (0.2 μM) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some intermediates (phenylpyruvate, 2-oxo-isocaproate, 3-hydroxy-butyryl-CoA, crotonyl-CoA) accumulated, while end products like isocaproate and butyrate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were enhanced, most likely to replace the missing ferredoxins. Proline reductase (prd), the corresponding ion pumping RNF complex (rnf) and the reaction product 5-aminovalerate were significantly enhanced. An ATP forming ATPase (atpCDGAHFEB) of the F0F1-type was induced while the formation of a ATP-consuming, proton-pumping V-type ATPase (atpDBAFCEKI) was decreased. The [Fe-S] enzyme-dependent pyruvate formate lyase (pfl), formate dehydrogenase (fdh) and hydrogenase (hyd) branch of glucose utilization and glycogen biosynthesis (glg) were significantly reduced, leading to an accumulation of glucose and pyruvate. The formation of [Fe-S] enzyme carbon monoxide dehydrogenase (coo) was inhibited. The fur mutant showed an increased sensitivity to vancomycin and polymyxin B. An intensive remodeling of the cell wall was observed, Polyamine biosynthesis (spe) was induced leading to an accumulation of spermine, spermidine, and putrescine. The fur mutant lost most of its flagella and motility. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of around 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, a significant replacement of iron requiring metabolic pathways and the restructuring of the cell surface for protection during the complex adaptation phase and was only partly directly regulated by Fur.

Manipulating Metabolic Plasticity By Targeting Pyruvate Kinase M2 in Platelets Inhibits Arterial Thrombosis

Background: Most of the cellular responses initiated upon platelet activation are energy consuming. Like normal cells, resting platelets rely primarily on oxidative phosphorylation (OXPHOS) to generate ATP, whereas activated platelets exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen, a phenomenon referred to as the Warburg effect in tumor cells) suggesting that metabolic plasticity exists in activated platelets. Although aerobic glycolysis yields less total ATP when compared to OXPHOS, the rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which is well suited for high-energy demands during platelet activation. Pyruvate kinases (PKs) catalyzes the final step of glycolysis, the formation of pyruvate and ATP from phosphoenolpyruvate and ADP. Four PK isoforms exist in mammals: L and R isoforms are expressed in the liver and red blood cells; the M1 isoform is expressed in most adult tissues that have high catabolic demands including muscle and brain; M2 is expressed in cells including activated platelets and leukocytes. While PKM1 and tetrameric PKM2 favor ATP production from OXPHOS through the TCA cycle, dimeric PKM2 drives aerobic glycolysis.Objective: We tested an innovative concept that by manipulating the energy demand of activated platelets (metabolic plasticity), by targeting PKM2, will inhibit platelet function and thrombosis.Methods: Using a specific inhibitor of PKM2 (inhibits PKM2 dimerization and stabilizes tetramers) and standardized platelet in vitro assays, we determined the mechanistic role of PKM2 in modulating platelet function in human and mice. To provide definitive evidence, we generated a megakaryocyte or platelet-specific PKM2-/- mouse (PKM2fl/flPF4Cre). Lactate assay was performed in WT and PKM2 null platelets. Susceptibility to thrombosis was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid artery thrombosis and laser injury models) by utilizing intravital microscopy.Results: We found that PKM2 is relatively highly expressed compared to PKM1 in human and murine platelets. Transmission electron microscopy (immunogold staining) revealed that PKM2 is found in the cytoplasm and a- granule in resting platelets, whereas most of PKM2 translocated to cytoplasm upon activation. Human and mouse platelets pretreated with PKM2 inhibitor exhibited decreased platelet aggregation to sub-optimal doses of collagen and convulxin but not to thrombin. In microfluidics flow chamber assay, human and whole mouse blood pretreated with PKM2 inhibitor formed small thrombi when perfused over collagen for 5 min at an arterial shear rate of 1500s-1 (P<0.05 vs. vehicle control). Platelets from PKM2fl/flPF4Cre mice exhibited decreased platelet aggregation to sub-optimal doses of collagen and convulxin, but not to thrombin, compared to PKM2fl/fl mice concomitant with decrease lactate production. In microfluidics flow chamber assay, whole blood from PKM2fl/flPF4Cre mice formed smaller thrombi when perfused over collagen for 5 min at an arterial shear rate of 1500s-1, compared to PKM2fl/fl mice. PKM2fl/flPF4Cre mice were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury-induced mesenteric artery thrombosis models (P<0.05 vs. vehicle control, N=10 mice/group), without altering hemostasis. PKM2 regulates the phosphorylation signal transducer and activator of transcription 3 (STAT3) and p-STAT3 act as a protein scaffold that facilitates the catalytic process of activating PLCg by kinase Syk in response to low-doses of collagen and CRP, but not TRAP or ADP in human and murine platelets. Interestingly, we found that PKM2 and STAT3 colocalized in the convulixn- stimulated control platelets and less phosphorylation of STAT-3 was observed in activated PKM2 null platelets (P<0.05 vs. WT), suggesting a non-glycolytic role of the PKM2 in regulating collagen signaling.Conclusions: Our results suggest that dimeric PKM2 regulates platelet function and arterial thrombosis most likely via GPVI signaling pathway. We suggest that manipulating metabolic plasticity by targeting dimeric PKM2 may be explored as a novel strategy to inhibit platelet function and arterial thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Phosphate and carbohydrate facilitate the formation of filamentous Salmonella enterica during osmotic stress.

Salmonella enterica is a human pathogen that can produce filamentous cells in response to environmental stress. The molecular mediators and biosynthetic pathways that contribute to the formation of filamentous cells (>10 µm in length) during osmotic stress are mostly unknown. The comparison of filamentous and non-filamentous cells in this study was aided by the use of a filtration step to separate cell types. Osmotic stress caused an efflux of phosphate from cells, and the addition of phosphate and a carbohydrate to Luria broth with 7 % NaCl (LB-7NaCl) significantly increased the proportion of filamentous cells in the population (58 %). In addition to direct measurements of intracellular and extracellular phosphate concentrations, the relative abundance of the iraP transcript that is induced by phosphate limitation was monitored. Non-filamentous cells had a greater relative abundance of iraP transcript than filamentous cells. IraP also affects the stability of RpoS, which regulates the general stress regulon, and was detected in non-filamentous cells but not filamentous cells. Markers of metabolic pathways for the production of acetyl-CoA (pflB, encoding for pyruvate formate lyase) and fatty acids (fabH) that are essential to membrane biosynthesis were found in greater abundance in filamentous cells than non-filamentous cells. There were no differences in the DNA, protein and biomass levels in filamentous and non-filamentous cells after 48 h of incubation, although the filamentous cells produced significantly (P<0.05) more acetate. This study found that phosphate and carbohydrate enhanced the formation of filamentous cells during osmotic stress, and there were differences in key regulatory elements and markers of metabolic pathways in filamentous and non-filamentous S. enterica.

The crystal structure of Proteus vulgaris tryptophan indole-lyase complexed with oxindolyl-L-alanine: implications for the reaction mechanism.

Tryptophan indole-lyase (TIL) is a bacterial enzyme which catalyzes the reversible formation of indole and ammonium pyruvate from L-tryptophan. Oxindolyl-L-alanine (OIA) is an inhibitor of TIL, with a Ki value of about 5 µM. The crystal structure of the complex of Proteus vulgaris TIL with OIA has now been determined at 2.1 Å resolution. The ligand forms a closed quinonoid complex with the pyridoxal 5'-phosphate (PLP) cofactor. The small domain rotates about 10° to close the active site, bringing His458 into position to donate a hydrogen bond to Asp133, which also accepts a hydrogen bond from the heterocyclic NH of the inhibitor. This brings Phe37 and Phe459 into van der Waals contact with the aromatic ring of OIA. Mutation of the homologous Phe464 in Escherichia coli TIL to Ala results in a 500-fold decrease in kcat/Km for L-tryptophan, with less effect on the reaction of other nonphysiological β-elimination substrates. Stopped-flow kinetic experiments of F464A TIL show that the mutation has no effect on the formation of quinonoid intermediates. An aminoacrylate intermediate is observed in the reaction of F464A TIL with S-ethyl-L-cysteine and benzimidazole. A model of the L-tryptophan quinonoid complex with PLP in the active site of P. vulgaris TIL shows that there would be a severe clash of Phe459 (∼1.5 Å apart) and Phe37 (∼2 Å apart) with the benzene ring of the substrate. It is proposed that this creates distortion of the substrate aromatic ring out of plane and moves the substrate upwards on the reaction coordinate towards the transition state, thus reducing the activation energy and accelerating the enzymatic reaction.

Enhanced production of succinic acid from methanol-organosolv pretreated Strophanthus preussii by recombinant Escherichia coli.

A biorefinery process for high yield production of succinic acid from biomass sugars was investigated using recombinant Escherichia coli. The major problem been addressed is utilization of waste biomass for the production of succinic acid using metabolic engineering strategy. Here, methanol extract of Strophanthus preussii was used for fermentation. The process parameters were optimized. Glucose (9g/L), galactose (4g/L), xylose (6g/L) and arabinose (0.5g/L) were the major sugars present in the methanol extract of S. preussii. E. coli K3OS with overexpression of soluble nucleotide pyridine transhydrogenase sthA and mutation of lactate dehydrogenase A (ldhA), phosphotransacetylase acetate kinase A (pta-ackA), pyruvate formate lyase B (pflB), pyruvate oxidase B (poxB), produced a final succinic acid concentration of 14.40g/L and yield of 1.10mol/mol total sugars after 72h dual-phase fermentation in M9 medium. Here, we show that the maximum theoretical yield using methanol extracts of S. preussii was 64%. Hence, methanol extract of S. preussii could be used for the production of biochemicals such as succinate, malate and pyruvate.

Artefactual formation of pyruvate from in-source conversion of lactate.

Lactate and pyruvate are high abundance products of glucose metabolism. Analysis of both molecules as part of metabolomics studies in cellular metabolism and physiology have been aided by advances in liquid chromatography-mass spectrometry (LC-MS). We used ion pairing-chromatography and negative ion mode ESI on an QExactive HF to perform stable isotope assisted metabolomics profiling of lactate and pyruvate metabolism. Using an LC-MS method for polar metabolite analysis we discovered an artefactual formation of pyruvate from in-source fragmentation of lactate. Surprisingly, this in-source fragmentation has not been previously described, thus we report this identification to warn other investigators. This artefact was detected by baseline chromatographic resolution of lactate and pyruvate by LC with confirmation of this artefact by stable isotope labeling of lactate and pyruvate. These findings have immediate implications for metabolomics studies by LC-MS and direct infusion MS, especially in negative ion mode, whereby users should resolve lactate from pyruvate or robustly quantify the potential formation of pyruvate from higher abundance lactate in their assays.

C-C Bond Formation of Mg- and Zn-Activated Carbon Dioxide.

Gas-phase activation of CO2 by chloride tagged metal atoms, [ClM]- (M=Mg, Zn), has been investigated by mass spectrometry and high-level quantum chemistry. Both metals activate CO2 with significant bending of the CO2 moiety to form complexes with the general formula [ClM,CO2 ]- . The structure of the metal-CO2 complex depends on the method of formation, and the energy landscapes and reaction dynamics have been probed by collisional induced dissociation and thermal ion molecule reactions with isotopically labeled species. Having established these structural relationships, the gas-phase reactivity of [ClM(κ2 -O2 C)]- with acetaldehyde (here considered a carbohydrate mimic) was then studied. Formation of lactate and enolate-pyruvate complexes are observed, showing that CO2 fixation by C-C bond formation takes place. For M=Zn, even formation of free pyruvate ([C3 H3 O3 ]- ) is observed. Implications of the observed CO2 reactivity for the electrochemical conversion of carbon dioxide, and to biochemical and artificial photosynthesis is briefly discussed. Detailed potential energy diagrams obtained by the quantum chemical calculations offer models consistent with experimental observation.

Unraveling the Mechanism of Cysteine Persulfide Formation Catalyzed by 3-Mercaptopyruvate Sulfurtransferases

Sulfhydration of reactive cysteines in target proteins is now recognized as a major route by which H2S mediates signal transduction and regulates various cellular processes. Among the enzymatic systems permitting the formation of cysteine persulfide from nonactivated sulfur compounds, 3-mercaptopyruvate sulfurtransferases can be considered as a model of thiolate-based chemistry for carbon–sulfur bond breaking. These ubiquitous enzymes transfer a sulfur atom from 3-mercaptopyruvate (3-MP) to a thiol acceptor via a cysteine-persulfide intermediate, but the mechanistic basis for its formation is still unclear. To address this question, kinetic approaches were developed for studying the reaction catalyzed by the human and Escherichia coli enzymes and the role of several conserved residues was also investigated. We showed that the first step of sulfur transfer that leads to pyruvate release and formation of the persulfide intermediate is very efficient for both enzymes. It critically depends on the electrostat...

Restricting glycolysis impairs brown adipocyte glucose and oxygen consumption.

During thermogenic activation, brown adipocytes take up large amounts of glucose. In addition, cold stimulation leads to an upregulation of glycolytic enzymes. Here we have investigated the importance of glycolysis for brown adipocyte glucose consumption and thermogenesis. Using siRNA-mediated knockdown in mature adipocytes, we explored the effect of glucose transporters and glycolytic enzymes on brown adipocyte functions such as consumption of glucose and oxygen. Basal oxygen consumption in brown adipocytes was equally dependent on glucose and fatty acid oxidation, whereas isoproterenol (ISO)-stimulated respiration was fueled mainly by fatty acids, with a significant contribution from glucose oxidation. Knockdown of glucose transporters in brown adipocytes not only impaired ISO-stimulated glycolytic flux but also oxygen consumption. Diminishing glycolytic flux by knockdown of the first and final enzyme of glycolysis, i.e., hexokinase 2 (HK2) and pyruvate kinase M (PKM), respectively, decreased glucose uptake and ISO-stimulated oxygen consumption. HK2 knockdown had a more severe effect, which, in contrast to PKM knockdown, could not be rescued by supplementation with pyruvate. Hence, brown adipocytes rely on glucose consumption and glycolytic flux to achieve maximum thermogenic output, with glycolysis likely supporting thermogenesis not only by pyruvate formation but also by supplying intermediates for efferent metabolic pathways.

Effects of glucose availability in Lactobacillus sakei; metabolic change and regulation of the proteome and transcriptome.

Effects of glucose availability were investigated in Lactobacillus sakei strains 23K and LS25 cultivated in anaerobic, glucose-limited chemostats set at high (D = 0.357 h-1) and low (D = 0.045 h-1) dilution rates. We observed for both strains a shift from homolactic towards more mixed acid fermentation when comparing high to low growth rates. However, this change was more pronounced for LS25 than for 23K, where dominating products were lactate>formate>acetate≥ethanol at both conditions. A multivariate approach was used for analyzing proteome and transcriptome data from the bacterial cultures, where the predictive power of the omics data was used for identifying features that can explain the differences in the end-product profiles. We show that the different degree of response to the same energy restriction revealed interesting strain specific regulation. An elevated formate production level during slow growth, more for LS25 than for 23K, was clearly reflected in correlating pyruvate formate lyase expression. With stronger effect for LS25, differential expression of the Rex transcriptional regulator and NADH oxidase, a target of Rex, indicated that maintainance of the cell redox balance, in terms of the NADH/NAD+ ratio, may be a key process during the metabolic change. The results provide a better understanding of different strategies that cells may deploy in response to changes in substrate availability.

Formic acid as a secondary substrate for succinic acid production by metabolically engineered Mannheimia succiniciproducens.

There has been much effort exerted to reduce one carbon (C1) gas emission to address climate change. As one promising way to more conveniently utilize C1 gas, several technologies have been developed to convert C1 gas into useful chemicals such as formic acid (FA). In this study, systems metabolic engineering was utilized to engineer Mannheimia succiniciproducens to efficiently utilize FA. 13 C isotope analysis of M. succiniciproducens showed that FA could be utilized through formate dehydrogenase (FDH) reaction and/or the reverse reaction of pyruvate formate lyase (PFL). However, the naturally favored forward reaction of PFL was found to lower the SA yield from FA. In addition, FA assimilation via FDH was found to be more efficient than the reverse reaction of PFL. Thus, the M. succiniciproducens LPK7 strain, which lacks in pfl, ldh, pta, and ack genes, was selected as a base strain. In silico metabolic analysis confirmed that utilization of FA would be beneficial for the enhanced production of SA and suggested FDH as an amplification target. To find a suitable FDH, four different FDHs from M. succiniciproducens, Methylobacterium extorquens, and Candida boidinii were amplified in LPK7 strain to enhance FA assimilation. High-inoculum density cultivation using 13 C labeled sodium formate was performed to evaluate FA assimilation efficiency. Fed-batch fermentations of the LPK7 (pMS3-fdh2 meq) strain was carried out using glucose, sucrose, or glycerol as a primary carbon source and FA as a secondary carbon source. As a result, this strain produced 76.11 g/L SA with the yield and productivity of 1.28 mol/mol and 4.08 g/L/h, respectively, using sucrose and FA as dual carbon sources. The strategy employed here will be similarly applicable in developing microorganisms to utilize FA and to produce valuable chemicals and materials from FA.

Integration between Glycolysis and Glutamate-Glutamine Cycle Flux May Explain Preferential Glycolytic Increase during Brain Activation, Requiring Glutamate.

The 1988 observation by Fox et al. (1988) that brief intense brain activation increases glycolysis (pyruvate formation from glucose) much more than oxidative metabolism has been abundantly confirmed. Specifically glycolytic increase was unexpected because the amount of ATP it generates is much smaller than that formed by subsequent oxidative metabolism of pyruvate. The present article shows that preferential glycolysis can be explained by metabolic processes associated with activation of the glutamate-glutamine cycle. The flux in this cycle, which is essential for production of transmitter glutamate and GABA, equals 75% of brain glucose utilization and each turn is associated with utilization of ~1 glucose molecule. About one half of the association between cycle flux and glucose metabolism occurs during neuronal conversion of glutamine to glutamate in a process similar to the malate-aspartate shuttle (MAS) except that glutamate is supplied from glutamine, not formed from α-ketoglutarate (αKG) as during operation of conventional MAS. Regular MAS function is triggered by one oxidative process in the cytosol during glycolysis causing NAD+ reduction to NADH. Since NADH cannot cross the mitochondrial membrane (MEM) for oxidation NAD+ is re-generated by conversion of cytosolic oxaloacetate (OAA) to malate, which enters the mitochondria for oxidation and in a cyclic process regenerates cytosolic OAA. Therefore MAS as well as the “pseudo-MAS” necessary for neuronal glutamate formation can only operate together with cytosolic reduction of NAD+ to NADH. The major process causing NAD+ reduction is glycolysis which therefore also must occur during neuronal conversion of glutamine to glutamate and may energize vesicular glutamate uptake which preferentially uses glycolytically derived energy. Another major contributor to the association between glutamate-glutamine cycle and glucose utilization is the need for astrocytic pyruvate to generate glutamate. Although some oxidative metabolism occurs during glutamate formation it is only one half of that during normal tricarboxylic acid (TCA) cycle function. Glutamate’s receptor stimulation leads to potassium ion (K+) release and astrocytic uptake, preferentially fueled by glycolysis and followed by release and neuronal re-accumulation. The activation-induced preferential glycolysis diminishes with continued activation and is followed by an increased ratio between oxidative metabolism and glycolysis, reflecting oxidation of generated glutamate and accumulated lactate.