Edwardsiella tarda is a gram-negative bacterium causing significant economic losses to aquaculture. E. tarda possesses NanA sialidase which removes sialic acids from α2–3 sialo-glycoprotein of host cells. However, the relationship between NanA sialidase activity and E. tarda invasiveness remains poorly understood. Furthermore, the pathway of sialic acid metabolism in E. tarda remains to be elucidated. We studied sialidase activity in several E. tarda strains and found that the pathogenic strains exhibited higher sialidase activity and greater up-regulation of the NanA mRNA level than non-pathogenic strain. Pathogenic strains also showed higher rates of infection in GAKS cells, and the infection was drastically suppressed by sialidase inhibitor. Additionally, NanA gene overexpression significantly increased infection and treatment of E. tarda with free sialic acid enhanced the rate of infection in GAKS cells. Sialic acid treatment enhanced mRNA levels of two N -acetylneuraminate lyases and one N -acetylneuraminate cytidylyltransferase. E. tarda uses sialic acid as a carbon source for growth via N -acetylneuraminate lyases. The strains with high N -acetylneuraminate cytidylyltransferase level showed greater sialylation of the lipopolysaccharides and glycoproteins. Our study establishes the significance of desialylation by E. tarda sialidase in the regulation of its invasiveness.

Plants have evolved the ability to derive the benzenoid moiety of the respiratory cofactor and antioxidant, ubiquinone (coenzyme Q), either from the b-oxidative metabolism of p -coumarate or from the peroxidative cleavage of kaempferol. Here, isotopic feeding assays, gene coexpression analysis and reverse genetics identified Arabidopsis 4-COUMARATE-COA LIGASE 8 ( 4-CL8 ; At5g38120 ) as a contributor to the b-oxidation of p -coumarate for ubiquinone biosynthesis. The enzyme is part of the same clade (V) of acyl-activating enzymes than At4g19010, a p -coumarate CoA ligase known to play a central role in the conversion of p -coumarate into 4-hydroxybenzoate. A 4-cl8 T-DNA knockout displayed a 20% decrease in ubiquinone content compared with wild-type plants, while 4-CL8 overexpression boosted ubiquinone content up to 150% of the control level.Similarly, the isotopic enrichment of ubiquinone's ring was decreased by 28% in the 4-cl8 knockout as compared to wild-type controls when Phe-[ Ring- 13 C 6 ] was fed to the plants. This metabolic blockage could be bypassed via the exogenous supply of 4-hydroxybenzoate, the product of p -coumarateb-oxidation. Arabidopsis4-CL8 displays a canonical peroxisomal targeting sequence type 1, and confocal microscopy experiments using fused fluorescent reporters demonstrated that this enzyme is imported into peroxisomes. Time course feeding assays using Phe-[ Ring- 13 C 6 ] in a series of Arabidopsis single and double knockouts blocked in the b-oxidative metabolism of p -coumarate ( 4-cl8; at4g19010; at4g19010 x 4-cl8 ) , flavonol biosynthesis ( flavanone-3-hydroxylase ), or both ( at4g19010 x flavanone-3-hydroxylase ) indicated that continuous high light treatments (500 µE m -2 s -1 ; 24h) markedly stimulated the de novo biosynthesis of ubiquinone independently of kaempferol catabolism.

Key physiological differences between bacterial and mammalian metabolism provide opportunities for the development of novel antimicrobials. We examined the role of the multifunctional enzyme S-adenosylhomocysteine/Methylthioadenosine (SAH/MTA) nucleosidase (Pfs) in the virulence of S. enterica var Typhimurium ( S. Typhimurium) in mice, using a defined Pfs deletion mutant (i.e. Δpfs ). Pfs was essential for growth of S. Typhimurium in M9 minimal medium, in tissue cultured cells, and in mice. Studies to resolve which of the three known functions of Pfs were key to murine virulence suggested that downstream production of auto-inducer 2, spermidine and methylthioribose were non-essential for Salmonella virulence in a highly sensitive murine model. Mass spectrometry revealed the accumulation of SAH in S. Typhimurium Δpfs and complementation of the Pfs mutant with the specific SAH hydrolase from Legionella pneumophila reduced SAH levels, fully restored growth ex vivo and the virulence of S. Typhimurium Δpfs for mice. The data suggest that Pfs may be a legitimate target for antimicrobial development, and that the key role of Pfs in bacterial virulence may be in reducing the toxic accumulation of SAH which, in turn, suppresses an undefined methyltransferase.

6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P) + by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5′-phosphate. PNPOs from Escherichia coli , Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this ‘side-activity’ is conserved. Remarkably, ‘pyridoxamine-phosphate oxidase-related proteins’ (PNPO-RP) from Nostoc punctiforme , A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5′-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH 3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH 3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).

ATP-dependent phospho enol pyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C 4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO 2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C 3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana : Ath PEPCK1 and Ath PEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. Ath PEPCK1 and Ath PEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited Ath PEPCK1 but had no effect on Ath PEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phospho enol pyruvate are targets of fine allosteric regulation.

Elevated serum homocysteine, an intermediate of cellular one-carbon metabolism, is an independent risk factor for cardiovascular disease (CVD). Folate deficiency increases serum homocysteine and may contribute to CVD progression. Vascular smooth muscle cells (VSMCs) regulate vascular contractility, but also contribute to repair processes in response to vascular injury. Nutritional deficiencies, like folate deficiency, are thought to impact on this phenotypic plasticity, possibly by epigenetic mechanisms. We have investigated the effect of folate deficiency on VSMCs in two cell culture systems representing early and late stages of smooth muscle cells differentiation. We find that folate deficiency promotes differentiation towards a more contractile phenotype as indicated by increased expression of respective marker genes. However, microarray analysis identified markers of striated muscle as the predominant gene expression change elicited by folate deficiency. These changes are not merely a reflection of cell cycle arrest, as foetal calf serum restriction or iron deficiency do not replicate the gene expression changes observed in response to folate deficiency. Folate deficiency only has a marginal effect on global DNA methylation. DNA methylation of CpG islands associated with genes regulated by folate deficiency remains unaffected. This supports our earlier findings in a mouse model system which also did not show any changes in global DNA methylation in response to folate and vitamin B6/B12 deficiency. These data suggest that folate deficiency enhances the expression of smooth muscle marker gene expression, promotes a shift towards a skeletal muscle phenotype, and does not regulate gene expression via DNA methylation.

Coxiella burnetii is a Gram-negative bacterium which causes Q fever, a complex and life-threatening infection with both acute and chronic presentations. C. burnetii invades a variety of host cell types and replicates within a unique vacuole derived from the host cell lysosome. In order to understand how C. burnetii survives within this intracellular niche, we have investigated the carbon metabolism of both intracellular and axenically cultivated bacteria. Both bacterial populations were shown to assimilate exogenous [ 13 C]glucose or [ 13 C]glutamate, with concomitant labeling of intermediates in glycolysis and gluconeogenesis, and in the TCA cycle. Significantly, the two populations displayed metabolic pathway profiles reflective of the nutrient availabilities within their propagated environments. Disruption of the C. burnetii glucose transporter, CBU0265, by transposon mutagenesis led to a significant decrease in [ 13 C]glucose utilization but did not abolish glucose usage, suggesting that C. burnetii express additional hexose transporters which may be able to compensate for the loss of CBU0265. This was supported by intracellular infection of human cells and in vivo studies in the insect model showing loss of CBU0265 had no impact on intracellular replication or virulence. Using this mutagenesis and [ 13 C]glucose labeling approach, we identified a second glucose transporter, CBU0347, the disruption of which also showed significant decreases in 13 C-label incorporation but did not impact intracellular replication or virulence. Together, these analyses indicate that C. burnetii may use multiple carbon sources in vivo and exhibits greater metabolic flexibility than expected.

Control of fatty acid storage and release in adipose tissue is fundamental in energy homeostasis and the development of obesity and type 2 diabetes. We here take the whole signalling network into account to identify how insulin and β-adrenergic stimulation in concert controls lipolysis in mature subcutaneous adipocytes obtained from non-diabetic and, in parallel, type 2 diabetic women. We report that, and show how, the anti-lipolytic effect of insulin can be fully explained by protein kinase B (PKB/Akt)-dependent activation of the phosphodiesterase PDE3B. Through the same PKB-dependent pathway β-adrenergic receptor signalling, via cAMP and PI3Kα, is anti-lipolytic and inhibits its own stimulation of lipolysis by 50%. Through this pathway both insulin and β-adrenergic signalling control phosphorylation of FOXO1. The dose–response of lipolysis is bell-shaped, such that insulin is anti-lipolytic at low concentrations, but at higher concentrations of insulin lipolysis was increasingly restored due to inhibition of PDE3B. The control of lipolysis was not altered in adipocytes from diabetic individuals. However, the release of fatty acids was increased by 50% in diabetes due to reduced reesterification of lipolytically liberated fatty acids. In conclusion, our results reveal mechanisms of control by insulin and β-adrenergic stimulation — in human adipocytes — that define a network of checks and balances ensuring robust control to secure uninterrupted supply of fatty acids without reaching concentrations that put cellular integrity at risk. Moreover, our results define how selective insulin resistance leave lipolytic control by insulin unaltered in diabetes, while the fatty acid release is substantially increased.

Fast and uniform germination is key to agricultural production and can be achieved by seed ‘priming’ techniques. Here, we characterised the responses of bread wheat ( Triticum aestivum L.) seeds to a hot steam treatment (‘BioFlash’), which accelerated water uptake, resulting in faster germination and seedling growth, typical traits of primed seed. Before the completion of germination, metabolite profiling of seeds revealed advanced accumulation of several amino acids (especially cysteine and serine), sugars (ribose, glucose), and organic acids (glycerate, succinate) in hot steam-treated seeds, whereas sugar alcohols (e.g. arabitol, mannitol) and trehalose decreased in all seeds. Tocochromanols (the ‘vitamin E family’) rose independently of the hot steam treatment. We further assessed shifts in the half-cell reduction potentials of low-molecular-weight (LMW) thiol-disulfide redox couples [i.e. glutathione disulfide (GSSG)/glutathione (GSH) and cystine/cysteine], alongside the activities of the reactive oxygen species (ROS)-processing enzyme superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase. Upon the first 4 h of imbibition, a rapid conversion of LMW disulfides to thiols occurred. Completion of germination was associated with a re-oxidation of the LMW thiol-disulfide cellular redox environment, before more reducing conditions were re-established during seedling growth, accompanied by an increase in all ROS-processing enzyme activities. Furthermore, changes in the thiol-disulfide cellular redox state were associated to specific stages of wheat seed germination. In conclusion, the priming effect of the hot steam treatment advanced the onset of seed metabolism, including redox shifts associated with germination and seedling growth.

The structure and function of glucose transporters of the mammalian GLUT family of proteins has been studied over many decades, and the proteins have fascinated numerous research groups over this time. This interest is related to the importance of the GLUTs as archetypical membrane transport facilitators, as key limiters of the supply of glucose to cell metabolism, as targets of cell insulin and exercise signalling and of regulated membrane traffic, and as potential drug targets to combat cancer and metabolic diseases such as type 2 diabetes and obesity. This review focusses on the use of chemical biology approaches and sugar analogue probes to study these important proteins.

Obesity stands as one of the greatest healthcare challenges of the 21 st century. Obesity in reproductive-age men is ever more frequent and is reaching upsetting levels. At the same time, fertility has taken an inverse direction and is decreasing, leading to an increased demand for fertility treatments. In half of infertile couples, there is a male factor alone or combined with a female factor. Furthermore, male fertility parameters such as sperm count and concentration went on a downward spiral during the last few decades and are now approaching the minimum levels established to achieve successful fertilization. Hence, the hypothesis that obesity and deleterious effects in male reproductive health, as reflected in deterioration of sperm parameters, are somehow related is tempting. Most often, overweight and obese individuals present leptin levels directly proportional to the increased fat mass. Leptin, besides the well-described central hypothalamic effects, also acts in several peripheral organs, including the testes, thus highlighting a possible regulatory role in male reproductive function. In the last years, research focusing on leptin effects in male reproductive function has unveiled additional roles and molecular mechanisms of action for this hormone at the testicular level. Herein, we summarize the novel molecular signals linking metabolism and male reproductive function with a focus on leptin signaling, mitochondria and relevant pathways for the nutritional support of spermatogenesis.

Whereas enzymes in the fumarylacetoacetate hydrolase (FAH) superfamily catalyze several distinct chemical reactions, the structural basis for their multi-functionality remains elusive. As a well-studied example, human FAH domain-containing protein 1 (FAHD1) is a mitochondrial protein displaying both acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. As mitochondrial ODx, FAHD1 acts antagonistically to pyruvate carboxylase, a key metabolic enzyme. Despite its importance for mitochondrial function, very little is known about the catalytic mechanisms underlying FAHD1 enzymatic activities, and the architecture of its ligated active site is currently ill defined. We present crystallographic data of human FAHD1 that provide new insights into the structure of the catalytic center at high resolution, featuring a flexible ‘lid’-like helical region which folds into a helical structure upon binding of the ODx inhibitor oxalate. The oxalate-driven structural transition results in the generation of a potential catalytic triad consisting of E33, H30 and an associated water molecule. In silico docking studies indicate that the substrate is further stabilized by a complex hydrogen-bond network, involving amino acids Q109 and K123, identified herein as potential key residues for FAHD1 catalytic activity. Mutation of amino acids H30, E33 and K123 each had discernible influence on the ApH and/or ODx activity of FAHD1, suggesting distinct catalytic mechanisms for both activities. The structural analysis presented here provides a defined structural map of the active site of FAHD1 and contributes to a better understanding of the FAH superfamily of enzymes.

l -Ascorbate, dehydro- l -ascorbic acid (DHA), and 2,3-diketo- l -gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (H 2 O 2 ), ‘superoxide’ (a ∼50 : 50 mixture of and ), hydroxyl radicals ( • OH, formed in Fenton mixtures), and illuminated riboflavin (generating singlet oxygen, 1 O 2 ). Products were monitored electrophoretically. DHA quenched H 2 O 2 far more effectively than superoxide, but the main products in both cases were 4- O -oxalyl- l -threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. H 2 O 2 , but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical • OH yield; it yielded oxalate, threonate, and OxT. 1 O 2 had no effect on DHA. DKG was oxidatively decarboxylated by H 2 O 2 , Fenton mixture, and 1 O 2 , forming a newly characterised product, 2-oxo- l - threo -pentonate (OTP; ‘2-keto- l -xylonate’). Superoxide yielded negligible OTP. Prolonged H 2 O 2 treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by H 2 O 2 , Fenton mixture, or 1 O 2 also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by H 2 O 2 and superoxide; DKG more readily by 1 O 2 . The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product ‘fingerprints’ are analytically useful, indicating which ROS are acting in vivo . l -Ascorbate, dehydro- l -ascorbic acid (DHA), and 2,3-diketo- l -gulonate (DKG) can all quench reactive oxygen species (ROS) in plants and animals. The vitamin C oxidation products thereby formed are investigated here. DHA and DKG were incubated aerobically at pH 4.7 with peroxide (H 2 O 2 ), ‘superoxide’ (a ∼50 : 50 mixture of and ), hydroxyl radicals ( • OH, formed in Fenton mixtures), and illuminated riboflavin (generating singlet oxygen, 1 O 2 ). Products were monitored electrophoretically. DHA quenched H 2 O 2 far more effectively than superoxide, but the main products in both cases were 4- O -oxalyl- l -threonate (4-OxT) and smaller amounts of 3-OxT and OxA + threonate. H 2 O 2 , but not superoxide, also yielded cyclic-OxT. Dilute Fenton mixture almost completely oxidised a 50-fold excess of DHA, indicating that it generated oxidant(s) greatly exceeding the theoretical • OH yield; it yielded oxalate, threonate, and OxT. 1 O 2 had no effect on DHA. DKG was oxidatively decarboxylated by H 2 O 2 , Fenton mixture, and 1 O 2 , forming a newly characterised product, 2-oxo- l - threo -pentonate (OTP; ‘2-keto- l -xylonate’). Superoxide yielded negligible OTP. Prolonged H 2 O 2 treatment oxidatively decarboxylated OTP to threonate. Oxidation of DKG by H 2 O 2 , Fenton mixture, or 1 O 2 also gave traces of 4-OxT but no detectable 3-OxT or cyclic-OxT. In conclusion, DHA and DKG yield different oxidation products when attacked by different ROS. DHA is more readily oxidised by H 2 O 2 and superoxide; DKG more readily by 1 O 2 . The diverse products are potential signals, enabling organisms to respond appropriately to diverse stresses. Also, the reaction-product ‘fingerprints’ are analytically useful, indicating which ROS are acting in vivo .

We show here that the M2 isoform of human pyruvate kinase (M2PYK) is susceptible to nitrosation and oxidation, and that these modifications regulate enzyme activity by preventing the formation of the active tetrameric form. The biotin-switch assay carried out on M1 and M2 isoforms showed that M2PYK is sensitive to nitrosation and that Cys326 is highly susceptible to redox modification. Structural and enzymatic studies have been carried out on point mutants for three cysteine residues (Cys424, Cys358, and Cys326) to characterise their potential roles in redox regulation. Nine cysteines are conserved between M2PYK and M1PYK. Cys424 is the only cysteine unique to M2PYK. C424S, C424A, and C424L showed a moderate effect on enzyme activity with 80, 100, and 140% activity, respectively, compared with M2PYK. C358 had been previously identified from in vivo studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK, suggesting that the in vivo resistance to oxidation for the Cys358Ser mutation is due to stabilisation of the tetrameric form of the enzyme. In contrast, the Cys326Ser mutant exists predominantly in monomeric form. A biotin-switch assay using this mutant also showed a significant reduction in biotinylation of M2PYK, confirming that this is a major target for nitrosation and probably oxidation. Our results show that the sensitivity of M2PYK to oxidation and nitrosation is regulated by its monomer–tetramer equilibrium. In the monomer state, residues (in particular C326) are exposed to oxidative modifications that prevent reformation of the active tetrameric form.

The mechanisms regulating oxidative phosphorylation during exercise remain poorly defined; however, key mitochondrial proteins, including carnitine palmitoyltransferase-I (CPT-I) and adenine nucleotide translocase, have redox-sensitive sites. Interestingly, muscle contraction has recently been shown to increase mitochondrial membrane potential and reactive oxygen species (ROS) production; therefore, we aimed to determine if mitochondrial-derived ROS influences bioenergetic responses to exercise. Specifically, we examined the influence of acute exercise on mitochondrial bioenergetics in WT (wild type) and transgenic mice (MCAT, mitochondrial-targeted catalase transgenic) possessing attenuated mitochondrial ROS. We found that ablating mitochondrial ROS did not alter palmitoyl-CoA (P-CoA) respiratory kinetics or influence the exercise-mediated reductions in malonyl CoA sensitivity, suggesting that mitochondrial ROS does not regulate CPT-I. In contrast, while mitochondrial protein content, maximal coupled respiration, and ADP (adenosine diphosphate) sensitivity in resting muscle were unchanged in the absence of mitochondrial ROS, exercise increased the apparent ADP K m (decreased ADP sensitivity) ∼30% only in WT mice. Moreover, while the presence of P-CoA decreased ADP sensitivity, it did not influence the basic response to exercise, as the apparent ADP K m was increased only in the presence of mitochondrial ROS. This basic pattern was also mirrored in the ability of ADP to suppress mitochondrial H 2 O 2 emission rates, as exercise decreased the suppression of H 2 O 2 only in WT mice. Altogether, these data demonstrate that while exercise-induced mitochondrial-derived ROS does not influence CPT-I substrate sensitivity, it inhibits ADP sensitivity independent of P-CoA. These data implicate mitochondrial redox signaling as a regulator of oxidative phosphorylation.

Fibroblast growth factors (FGF) 19, 21 and 23 are characterized by being endocrinely secreted and require co-receptor α-klotho or β-klotho (BKL) for binding and activation of the FGF receptors (FGFR). FGF15 is the rodent orthologue of human FGF19, but the two proteins share only 52% amino acid identity. Despite the physiological role of FGF21 and FGF19 being quite different, both lower blood glucose (BG) when administered to diabetic mice. The present study was designed to clarify why two human proteins with distinct physiological functions both lower BG in db/db mice and if the mouse orthologue FGF15 has similar effect to FGF19 and FGF21. Recombinant human FGF19, -21 and a mouse FGF15 variant (C110S) were expressed and purified from Escherichia coli . While rhFGF19 (recombinant human fibroblast growth factor 19) and rhFGF21 (recombinant human fibroblast growth factor) bound FGFRs in complex with both human and mouse BKL, rmFGF15CS (recombinant mouse fibroblast growth factor 15 C110S) only bound the FGFRs when combined with mouse BKL. Recombinant hFGF21 and rhFGF19, but not rmFGF15CS, increased glucose uptake in mouse adipocytes, while rhFGF19 and rmFGF15CS potently decreased Cyp7a1 expression in rat hepatocytes. The lack of effect of rmFGF15CS on glucose uptake in adipocytes was associated with rmFGF15CS's inability to signal through the FGFR1c/mouse BKL complex. In db/db mice, only rhFGF19 and rhFGF21 decreased BG while rmFGF15CS and rhFGF19, but not rhFGF21, increased total cholesterol. These data demonstrate receptor- and species-specific differential activity of FGF15 and FGF19 which should be taken into consideration when FGF19 is used as a substitute for FGF15.

Although iron and copper are co-ordinately regulated in living cells, the homeostatic effects of each of these metals on the other remain unknown. Here, we show the function of AfMac1, a transcriptional activator of the copper and iron regulons of Aspergillus fumigatus , on the interaction between iron and copper. In addition to the copper-specific AfMac1-binding motif 5′-TGT GCTCA -3′ found in the promoter region of ctrC , the iron-specific AfMac1-binding motif 5′- AT (C/G)NN(A/T) T (A/C)-3′ was identified in the iron regulon but not in the copper regulon by ChIP sequence analysis. Furthermore, mutation of the AfMac1-binding motif of sit1 eliminated AfMac1-mediated sit1 up-regulation. Interestingly, the regulation of gene expression in the iron regulon by AfMac1 was not affected by copper and vice versa . AfMac1 localized to the nucleus under iron- or copper-depleted conditions, and AfMac1 was mostly detected in the cytoplasm under iron- or copper-replete conditions . Taken together, these results suggest that A. fumigatus independently regulates iron and copper homeostasis in a manner that involves AfMac1 and mutual interactions.

In the first committed step of histidine biosynthesis, adenosine 5′-triphosphate (ATP) and 5-phosphoribosyl-α1-pyrophosphate (PRPP), in the presence of ATP phosphoribosyltransferase (ATP-PRT, EC 2.4.2.17), yield phosphoribosyl-ATP. ATP-PRTs are subject to feedback inhibition by histidine that allosterically binds between the regulatory domains. Histidine biosynthetic pathways of bacteria, lower eukaryotes, and plants are considered promising targets for the design of antibiotics, antifungal agents, and herbicides because higher organisms are histidine heterotrophs. Plant ATP-PRTs are similar to one of the two types of their bacterial counterparts, the long-type ATP-PRTs. A biochemical and structural study of ATP-PRT from the model legume plant, Medicago truncatula ( Medtr ATP-PRT1) is reported herein. Two crystal structures, presenting homohexameric Medtr ATP-PRT1 in its relaxed (R-) and histidine-bound, tense (T-) states allowed to observe key features of the enzyme and provided the first structural insights into an ATP-PRT from a eukaryotic organism. In particular, they show pronounced conformational reorganizations during R-state to T-state transition that involves substantial movements of domains. This rearrangement requires a trans - to cis - switch of a peptide backbone within the hinge region of Medtr ATP-PRT1. A C-terminal α-helix, absent in bacteria, reinforces the hinge that is constituted by two peptide strands. As a result, conformations of the R- and T-states are significantly different from the corresponding states of prokaryotic enzymes with known 3-D structures. Finally, adenosine 5′-monophosphate (AMP) bound at the active site is consistent with a competitive (and synergistic with histidine) nature of AMP inhibition.

Until recently, prebiotic precursors to metabolic pathways were not known. In parallel, chemistry achieved the synthesis of amino acids and nucleotides only in reaction sequences that do not resemble metabolic pathways, and by using condition step changes, incompatible with enzyme evolution. As a consequence, it was frequently assumed that the topological organisation of the metabolic pathway has formed in a Darwinian process. The situation changed with the discovery of a non-enzymatic glycolysis and pentose phosphate pathway. The suite of metabolism-like reactions is promoted by a metal cation, (Fe(II)), abundant in Archean sediment, and requires no condition step changes. Knowledge about metabolism-like reaction topologies has accumulated since, and supports non-enzymatic origins of gluconeogenesis, the S -adenosylmethionine pathway, the Krebs cycle, as well as CO 2 fixation. It now feels that it is only a question of time until essential parts of metabolism can be replicated non-enzymatically. Here, I review the ‘accidents’ that led to the discovery of the non-enzymatic glycolysis, and on the example of a chemical network based on hydrogen cyanide, I provide reasoning why metabolism-like non-enzymatic reaction topologies may have been missed for a long time. Finally, I discuss that, on the basis of non-enzymatic metabolism-like networks, one can elaborate stepwise scenarios for the origin of metabolic pathways, a situation that increasingly renders the origins of metabolism a tangible problem.

Trypanosomatid parasites are the infectious agents causing Chagas disease, visceral and cutaneous leishmaniasis and human African trypanosomiasis. Recent work of others has implicated an aldo-keto reductase (AKR) in the susceptibility and resistance of Trypanosoma cruzi to benznidazole, a drug used to treat Chagas disease. Here, we show that Tc AKR and homologues in the related parasites Trypanosoma brucei and Leishmania donovani do not reductively activate monocyclic (benznidazole, nifurtimox and fexinidazole) or bicyclic nitro-drugs such as PA-824. Rather, these enzymes metabolise a variety of toxic ketoaldehydes, such as glyoxal and methylglyoxal, suggesting a role in cellular defence against chemical stress. UPLC-QToF/MS analysis of benznidazole bioactivation by T. cruzi cell lysates confirms previous reports identifying numerous drug metabolites, including a dihydro-dihydroxy intermediate that can dissociate to form N -benzyl-2-guanidinoacetamide and glyoxal, a toxic DNA-glycating and cross-linking agent. Thus, we propose that Tc AKR contributes to benznidazole resistance by the removal of toxic glyoxal. In addition, three of the four enzymes studied here display activity as prostaglandin F 2α synthases, despite the fact that there are no credible cyclooxygenases in these parasites to account for formation of the precursor PGH 2 from arachidonic acid. Our studies suggest that arachidonic acid is first converted non-enzymatically in parasite lysates to (PGH 2 -like) regioisomers by free radical-mediated peroxidation and that AKRs convert these lipid peroxides into isoprostanes, including prostaglandin F 2α and 8-iso-prostaglandin F 2α .

Dihydroxyacetone (DHA) is the smallest ketotriose, and it is utilized by many organisms as an energy source. However, at higher concentrations, DHA becomes toxic towards several organisms including the budding yeast Saccharomyces cerevisiae . In the present study, we show that DHA toxicity is due to its spontaneous conversion to methylglyoxal (MG) within yeast cells. A mutant defective in MG-metabolizing enzymes ( glo1 Δ gre2 Δ gre3 Δ) exhibited higher susceptibility to DHA. Intracellular MG levels increased following the treatment of glo1 Δ gre2 Δ gre3 Δ cells with DHA. We previously reported that MG depolarized the actin cytoskeleton and changed vacuolar morphology. We herein demonstrated the depolarization of actin and morphological changes in vacuoles following a treatment with DHA. Furthermore, we found that both MG and DHA caused the morphological change in nucleus, and inhibited the nuclear division. Our results suggest that the conversion of DHA to MG is a dominant contributor to its cytotoxicity.

Intercellular communications play a major role in tissue homeostasis. In pathologies such as cancer, cellular interactions within the tumor microenvironment (TME) contribute to tumor progression and resistance to therapy. Tunneling nanotubes (TNTs) are newly discovered long-range intercellular connections that allow the exchange between cells of various cargos, ranging from ions to whole organelles such as mitochondria. TNT-transferred mitochondria were shown to change the metabolism and functional properties of recipient cells as reported for both normal and cancer cells. Metabolic plasticity is now considered a hallmark of cancer as it notably plays a pivotal role in drug resistance. The acquisition of cancer drug resistance was also associated to TNT-mediated mitochondria transfer, a finding that relates to the role of mitochondria as a hub for many metabolic pathways. In this review, we first give a brief overview of the various mechanisms of drug resistance and of the cellular communication means at play in the TME, with a special focus on the recently discovered TNTs. We further describe recent studies highlighting the role of the TNT-transferred mitochondria in acquired cancer cell drug resistance. We also present how changes in metabolic pathways, including glycolysis, pentose phosphate and lipid metabolism, are linked to cancer cell resistance to therapy. Finally, we provide examples of novel therapeutic strategies targeting mitochondria and cell metabolism as a way to circumvent cancer cell drug resistance.

In demyelinating nervous system disorders, myelin basic protein (MBP), a major component of the myelin sheath, is proteolyzed and its fragments are released in the neural environment. Here, we demonstrated that, in contrast with MBP, the cellular uptake of the cryptic 84–104 epitope (MBP84-104) did not involve the low-density lipoprotein receptor-related protein-1, a scavenger receptor. Our pull-down assay, mass spectrometry and molecular modeling studies suggested that, similar with many other unfolded and aberrant proteins and peptides, the internalized MBP84-104 was capable of binding to the voltage-dependent anion-selective channel-1 (VDAC-1), a mitochondrial porin. Molecular modeling suggested that MBP84-104 directly binds to the N-terminal α-helix located midway inside the 19 β-blade barrel of VDAC-1. These interactions may have affected the mitochondrial functions and energy metabolism in multiple cell types. Notably, MBP84-104 caused neither cell apoptosis nor affected the total cellular ATP levels, but repressed the aerobic glycolysis (lactic acid fermentation) and decreased the l -lactate/ d -glucose ratio (also termed as the Warburg effect) in normal and cancer cells. Overall, our findings implied that because of its interactions with VDAC-1, the cryptic MBP84-104 peptide invoked reprogramming of the cellular energy metabolism that favored enhanced cellular activity, rather than apoptotic cell death. We concluded that the released MBP84-104 peptide, internalized by the cells, contributes to the reprogramming of the energy-generating pathways in multiple cell types.

β-Ureidopropionase (βUP) catalyzes the third step of the reductive pyrimidine catabolic pathway responsible for breakdown of uracil-, thymine- and pyrimidine-based antimetabolites such as 5-fluorouracil. Nitrilase-like βUPs use a tetrad of conserved residues (Cys233, Lys196, Glu119 and Glu207) for catalysis and occur in a variety of oligomeric states. Positive co-operativity toward the substrate N -carbamoyl-β-alanine and an oligomerization-dependent mechanism of substrate activation and product inhibition have been reported for the enzymes from some species but not others. Here, the activity of recombinant human βUP is shown to be similarly regulated by substrate and product, but in a pH-dependent manner. Existing as a homodimer at pH 9, the enzyme increasingly associates to form octamers and larger oligomers with decreasing pH. Only at physiological pH is the enzyme responsive to effector binding, with N -carbamoyl-β-alanine causing association to more active higher molecular mass species, and β-alanine dissociation to inactive dimers. The parallel between the pH and ligand-induced effects suggests that protonation state changes play a crucial role in the allosteric regulation mechanism. Disruption of dimer–dimer interfaces by site-directed mutagenesis generated dimeric, inactive enzyme variants. The crystal structure of the T299C variant refined to 2.08 Å resolution revealed high structural conservation between human and fruit fly βUP, and supports the hypothesis that enzyme activation by oligomer assembly involves ordering of loop regions forming the entrance to the active site at the dimer–dimer interface, effectively positioning the catalytically important Glu207 in the active site.

Platelets (PLTs) deteriorate over time when stored within blood banks through a biological process known as PLT storage lesion (PSL). Here, we describe the refinement of the biochemical model of PLT metabolism, iAT-PLT-636, and its application to describe and investigate changes in metabolism during PLT storage. Changes in extracellular acetate and citrate were measured in buffy coat and apheresis PLT units over 10 days of storage in the PLT additive solution T-Sol. Metabolic network analysis of these data was performed alongside our prior metabolomics data to describe the metabolism of fresh (days 1–3), intermediate (days 4–6), and expired (days 7–10) PLTs. Changes in metabolism were studied by comparing metabolic model flux predictions of iAT-PLT-636 between stages and between collection methods. Extracellular acetate and glucose contribute most to central carbon metabolism in PLTs. The anticoagulant citrate is metabolized in apheresis-stored PLTs and is converted into aconitate and, to a lesser degree, malate. The consumption of nutrients changes during storage and reflects altered PLT activation profiles following their collection. Irrespective of the collection method, a slowdown in oxidative phosphorylation takes place, consistent with mitochondrial dysfunction during PSL. Finally, the main contributors to intracellular ammonium and NADPH are highlighted. Future optimization of flux through these pathways provides opportunities to address intracellular pH changes and reactive oxygen species, which are both of importance to PSL. The metabolic models provide descriptions of PLT metabolism at steady state and represent a platform for future PLT metabolic research.

Apolipoprotein M (apoM) is a carrier and a modulator of sphingosine 1-phosphate (S1P), an important multifunctional bioactive lipid. Since peroxisome proliferator-activated receptor γ (PPARγ) is reportedly associated with the function and metabolism of S1P, we investigated the modulation of apoM/S1P homeostasis by PPARγ. First, we investigated the modulation of apoM and S1P homeostasis by the overexpression or knockdown of PPARγ in HepG2 cells and found that both the overexpression and the knockdown of PPARγ decreased apoM expression and S1P synthesis. When we activated or suppressed the PPARγ more mildly with pioglitazone or GW9662, we found that pioglitazone suppressed apoM expression and S1P synthesis, while GW9662 increased them. Next, we overexpressed PPARγ in mouse liver through adenoviral gene transfer and observed that both the plasma and hepatic apoM levels and the plasma S1P levels decreased, while the hepatic S1P levels increased, in the presence of enhanced sphingosine kinase activity. Treatment with pioglitazone decreased both the plasma and hepatic apoM and S1P levels only in diet-induced obese mice. Moreover, the overexpression of apoM increased, while the knockdown of apoM suppressed PPARγ activities in HepG2 cells. These results suggested that PPARγ regulates the S1P levels by modulating apoM in a bell-shaped manner, with the greatest levels of apoM/S1P observed when PPARγ was mildly expressed and that hepatic apoM/PPARγ axis might maintain the homeostasis of S1P metabolism.

In all living organisms, coenzyme A (CoA) is an essential cofactor with a unique design allowing it to function as an acyl group carrier and a carbonyl-activating group in diverse biochemical reactions. It is synthesized in a highly conserved process in prokaryotes and eukaryotes that requires pantothenic acid (vitamin B5), cysteine and ATP. CoA and its thioester derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. A novel unconventional function of CoA in redox regulation has been recently discovered in mammalian cells and termed protein CoAlation. Here, we report for the first time that protein CoAlation occurs at a background level in exponentially growing bacteria and is strongly induced in response to oxidizing agents and metabolic stress. Over 12% of Staphylococcus aureus gene products were shown to be CoAlated in response to diamide-induced stress . In vitro CoAlation of S. aureus glyceraldehyde-3-phosphate dehydrogenase was found to inhibit its enzymatic activity and to protect the catalytic cysteine 151 from overoxidation by hydrogen peroxide. These findings suggest that in exponentially growing bacteria, CoA functions to generate metabolically active thioesters, while it also has the potential to act as a low-molecular-weight antioxidant in response to oxidative and metabolic stress.

We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that, within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors, while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer–monomer dissociation K d is estimated to be ∼0.9 µM with a slow dissociation rate ( t 1/2 ∼ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such ‘allostatic’ regulation may be important in metabolic reprogramming and influencing cell fate.

Metabolic syndrome comprises a series of health conditions, such as increased blood pressure, high blood sugar, excess abdominal fat, and altered circulating cholesterol or triglyceride levels. A fast growing number of affected individuals are at an increased risk of heart disease, stroke and type-2 diabetes. Obesity, especially build-up of visceral fat, is a recognized major risk factor for the development of metabolic syndrome. However, our understanding of the mechanistic links and biomarkers that associate visceral fat with the development of conditions underlying metabolic syndrome is still inadequate. In a recent paper published in the Biochemical Journal [ Biochem. J. (2018) 475, 1019–1035], Candi et al. address this lack of knowledge, performing high-throughput metabolomics analysis of visceral fat isolated from obese individuals, with and without metabolic syndrome, and non-obese healthy controls. The authors identify alterations in metabolic pathways that distinguish pathologically from healthy obese subjects. They identify metabolic cues that point to oxidative and inflammatory burden as the leitmotifs of metabolic syndrome. Of particular interest is the identification of increased metabolism of γ-glutamyl amino acids and plasmalogens in pathological obesity. γ-glutamyl amino acids, generated through the transfer of a γ-glutamyl moiety from glutathione to an amino acid acceptor, are involved in glutathione metabolism and the response to oxidative stress, whereas plasmalogens, a poorly studied class of phospholipids, are known contributors to insulin resistance and hypertension. Both classes of metabolites are intriguing candidate biomarkers that warrant further investigation.

The target of rapamycin (TOR) is a protein kinase that, by forming complexes with partner proteins, governs diverse cellular signalling networks to regulate a wide range of processes. TOR thus plays central roles in maintaining normal cellular functions and, when dysregulated, in diverse diseases. TOR forms two distinct types of multiprotein complexes (TOR complexes 1 and 2, TORC1 and TORC2). TORC1 and TORC2 differ in their composition, their control and their substrates, so that they play quite distinct roles in cellular physiology. Much effort has been focused on deciphering the detailed regulatory links within the TOR pathways and the structure and control of TOR complexes. In this review, we summarize recent advances in understanding mammalian (m) TORC2, its structure, its regulation, and its substrates, which link TORC2 signalling to the control of cell functions. It is now clear that TORC2 regulates several aspects of cell metabolism, including lipogenesis and glucose transport. It also regulates gene transcription, the cytoskeleton, and the activity of a subset of other protein kinases.

Mitochondrial pyruvate carrier (MPC), which is essential for mitochondrial pyruvate usage, mediates the transport of cytosolic pyruvate into mitochondria. Low MPC expression is associated with various cancers, and functionally associated with glycolytic metabolism and stemness. However, the mechanism by which MPC expression is regulated is largely unknown. In this study, we showed that MPC1 is down-regulated in human renal cell carcinoma (RCC) due to strong suppression of peroxisome proliferator-activated receptor-gamma co-activator (PGC)-1 alpha (PGC-1α). We also demonstrated that overexpression of PGC-1α stimulates MPC1 transcription, while depletion of PGC-1α by siRNA suppresses MPC expression. We found that PGC-1α interacts with estrogen-related receptor-alpha (ERR-α) and recruits it to the ERR-α response element motif located in the proximal MPC1 promoter, resulting in efficient activation of MPC1 expression. Furthermore, the MPC inhibitor, UK5099, blocked PGC-1α-induced pyruvate-dependent mitochondrial oxygen consumption. Taken together, our results suggest that MPC1 is a novel target gene of PGC-1α. In addition, low expression of PGC-1α in human RCC might contribute to the reduced expression of MPC, resulting in impaired mitochondrial respiratory capacity in RCC by limiting the transport of pyruvate into the mitochondrial matrix.

Phenotypic and functional heterogeneity is one of the most relevant features of cancer cells within different tumor types and is responsible for treatment failure. Cancer stem cells (CSCs) are a population of cells with stem cell-like properties that are considered to be the root cause of tumor heterogeneity, because of their ability to generate the full repertoire of cancer cell types. Moreover, CSCs have been invoked as the main drivers of metastatic dissemination and therapeutic resistance. As such, targeting CSCs may be a useful strategy to improve the effectiveness of classical anticancer therapies. Recently, metabolism has been considered as a relevant player in CSC biology, and indeed, oncogenic alterations trigger the metabolite-driven dissemination of CSCs. More interestingly, the action of metabolic pathways in CSC maintenance might not be merely a consequence of genomic alterations. Indeed, certain metabotypic phenotypes may play a causative role in maintaining the stem traits, acting as an orchestrator of stemness. Here, we review the current studies on the metabolic features of CSCs, focusing on the biochemical energy pathways involved in CSC maintenance and propagation. We provide a detailed overview of the plastic metabolic behavior of CSCs in response to microenvironment changes, genetic aberrations, and pharmacological stressors. In addition, we describe the potential of comprehensive metabolic approaches to identify and selectively eradicate CSCs, together with the possibility to ‘force’ CSCs within certain metabolic dependences, in order to effectively target such metabolic biochemical inflexibilities. Finally, we focus on targeting mitochondria to halt CSC dissemination and effectively eradicate cancer.

In eukaryotic cells, amino acid biosynthesis is feedback-inhibited by amino acids through inhibition of the conserved protein kinase Gcn2. This decreases phosphorylation of initiation factor eIF2α, resulting in general activation of translation but inhibition of translation of mRNA for transcription factor (TF) Gcn4 in yeast or ATF4 in mammals. These TFs are positive regulators of amino acid biosynthetic genes. As several enzymes of amino acid biosynthesis contain iron–sulfur clusters (ISCs) and iron excess is toxic, iron and amino acid homeostasis should be co-ordinated. Working with the yeast Saccharomyces cerevisiae , we found that amino acid supplementation down-regulates expression of genes for iron uptake and decreases intracellular iron content. This cross-regulation requires Aft1, the major TF activated by iron scarcity, as well as Gcn2 and phosphorylatable eIF2α but not Gcn4. A mutant with constitutive activity of Gcn2 ( GCN2 c ) shows less repression of iron transport genes by amino acids and increased nuclear localization of Aft1 in an iron-poor medium, and increases iron content in this medium. As Aft1 is activated by depletion of mitochondrial ISCs, it is plausible that the Gcn2–eIF2α pathway inhibits the formation of these complexes. Accordingly, the GCN2 c mutant has strongly reduced activity of succinate dehydrogenase, an iron–sulfur mitochondrial enzyme, and is unable to grow in media with very low iron or with galactose instead of glucose, conditions where formation of ISCs is specially needed. This mechanism adjusts the uptake of iron to the needs of amino acid biosynthesis and expands the list of Gcn4-independent activities of the Gcn2–eIF2α regulatory system.

Seed oil from flax ( Linum usitatissimum ) is enriched in α-linolenic acid (ALA; 18:3Δ 9 cis ,12 cis ,15 cis ), but the biochemical processes underlying the enrichment of flax seed oil with this polyunsaturated fatty acid are not fully elucidated. Here, a potential process involving the catalytic actions of long-chain acyl-CoA synthetase (LACS) and diacylglycerol acyltransferase (DGAT) is proposed for ALA enrichment in triacylglycerol (TAG). LACS catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which in turn may serve as an acyl-donor in the DGAT-catalyzed reaction leading to TAG. To test this hypothesis, flax LACS and DGAT cDNAs were functionally expressed in Saccharomyces cerevisiae strains to probe their possible involvement in the enrichment of TAG with ALA. Among the identified flax LACSs, LuLACS8A exhibited significantly enhanced specificity for ALA over oleic acid (18:1Δ 9 cis ) or linoleic acid (18:2Δ 9 cis ,12 cis ). Enhanced α-linolenoyl-CoA specificity was also observed in the enzymatic assay of flax DGAT2 (LuDGAT2-3), which displayed ∼20 times increased preference toward α-linolenoyl-CoA over oleoyl-CoA. Moreover, when LuLACS8A and LuDGAT2-3 were co-expressed in yeast, both in vitro and in vivo experiments indicated that the ALA-containing TAG enrichment process was operative between LuLACS8A- and LuDGAT2-3-catalyzed reactions. Overall, the results support the hypothesis that the cooperation between the reactions catalyzed by LACS8 and DGAT2 may represent a route to enrich ALA production in the flax seed oil.

Non-canonical d -amino acids play important roles in bacteria including control of peptidoglycan metabolism and biofilm disassembly. Bacteria appear to produce non-canonical d -amino acids to adapt to various environmental changes, and understanding the biosynthetic pathways is important. We identified novel amino acid racemases possessing the ability to produce non-canonical d -amino acids in Escherichia coli and Bacillus subtilis in our previous study, whereas the biosynthetic pathways of these d -amino acids still remain unclear. In the present study, we demonstrated that two cystathionine β-lyases (MetC and MalY) from E. coli produce non-canonical d -amino acids including non-proteinogenic amino acids. Furthermore, MetC displayed d - and l -serine (Ser) dehydratase activity. We characterised amino acid racemase, Ser dehydratase and cysteine lyase activities, and all were higher for MetC. Interestingly, all three activities were at a comparable level for MetC, although optimal conditions for each reaction were distinct. These results indicate that MetC and MalY are multifunctional enzymes involved in l -methionine metabolism and the production of d -amino acids, as well as d - and l -Ser metabolism. To our knowledge, this is the first evidence that cystathionine β-lyase is a multifunctional enzyme with three different activities.

Ca 2+ can be released from cell compartments to the cytosol during stress conditions. We discuss here the causes of Ca 2+ release under conditions of ATP concentration decline that result in the suppression of ATPases and activation of calcium ion channels. The main signaling and metabolic consequences of Ca 2+ release are considered for stressed plant cells. The signaling function includes generation and spreading of calcium waves, while the metabolic function results in the activation of particular enzymes and genes. Ca 2+ is involved in the activation of glutamate decarboxylase, initiating the γ-aminobutyric acid shunt and triggering the formation of alanine, processes which play a role, in particular, in pH regulation. Ca 2+ activates the transcription of several genes, e.g. of plant hemoglobin (phytoglobin, Pgb) which scavenges nitric oxide and regulates redox and energy balance through the Pgb–nitric oxide cycle. This cycle involves NADH and NADPH oxidation from the cytosolic side of mitochondria, in which Ca 2+ - and low pH-activated external NADH and NADPH dehydrogenases participate. Ca 2+ can also activate the genes of alcohol dehydrogenase and pyruvate decarboxylase stimulating hypoxic fermentation. It is concluded that calcium is a primary factor that causes the metabolic shift under conditions of oxygen deficiency.

Bacterial sulfoglycolytic pathways catabolize sulfoquinovose (SQ), or glycosides thereof, to generate a three-carbon metabolite for primary cellular metabolism and a three-carbon sulfonate that is expelled from the cell. Sulfoglycolytic operons encoding an Embden–Meyerhof–Parnas-like or Entner–Doudoroff (ED)-like pathway harbor an uncharacterized gene ( yihR in Escherichia coli ; PpSQ1_00415 in Pseudomonas putida ) that is up-regulated in the presence of SQ, has been annotated as an aldose-1-epimerase and which may encode an SQ mutarotase. Our sequence analyses and structural modeling confirmed that these proteins possess mutarotase-like active sites with conserved catalytic residues. We overexpressed the homolog from the sulfo-ED operon of Herbaspirillum seropedicaea ( Hs SQM) and used it to demonstrate SQ mutarotase activity for the first time. This was accomplished using nuclear magnetic resonance exchange spectroscopy, a method that allows the chemical exchange of magnetization between the two SQ anomers at equilibrium. Hs SQM also catalyzed the mutarotation of various aldohexoses with an equatorial 2-hydroxy group, including d -galactose, d -glucose, d -glucose-6-phosphate (Glc-6-P), and d -glucuronic acid, but not d -mannose. Hs SQM displayed only 5-fold selectivity in terms of efficiency ( k cat / K M ) for SQ versus the glycolysis intermediate Glc-6-P; however, its proficiency [ k uncat /( k cat / K M )] for SQ was 17 000-fold better than for Glc-6-P, revealing that Hs SQM preferentially stabilizes the SQ transition state.

Gentiobiose, a β-1,6-linked glycosyl-disaccharide, accumulates abundantly in Gentianaceae and is involved in aspects of plant development, such as fruits ripening and release of bud dormancy. However, the mechanisms regulating the amount of gentio-oligosaccharide accumulation in plants remain obscure. The present study aimed to identify an enzyme that modulates gentio-oligosaccharide amount in gentian ( Gentiana triflora ). A protein responsible for gentiobiose hydrolysis, GtGen3A, was identified by partial purification and its peptide sequence analysis. The enzyme had a molecular mass of ∼67 kDa without a secretory signal peptide sequence. Sequence analysis revealed that GtGen3A could be a β-glucosidase member belonging to glycoside hydrolase family 3 (GH3). GtGen3A showed a homology to GH3 β-glucan exohydrolases, ExoI of Hordeum vulgare , and ExgI from Zea mays , which preferentially hydrolyzed β-1,3- and β-1,4-linked oligosaccharides. The purified recombinant GtGen3A (rGtGen3A) produced in Escherichia coli showed optimal reaction at pH 6.5 and 20°C. The rGtGen3A liberated glucose from β-1,2-, β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and showed the highest activity toward gentiotriose among the substrates tested. Kinetic analysis also revealed that rGtGen3A preferentially hydrolyzed gentiotriose. Virus-induced gene silencing of Gtgen3A in gentian plantlets resulted in predominant accumulation of gentiotriose rather than gentiobiose. Furthermore, the expression level of Gtgen3A was almost similar to the amount of gentiobiose in field-grown gentians. These findings suggest that the main function of GtGen3A is the hydrolysis of gentiotriose to gentiobiose, and that GtGen3A plays a role in modulating gentiobiose amounts in gentian.

The movement of lipids across mitochondrial membranes represents a rate-limiting step in fatty acid oxidation within the heart. A key regulatory point in this process is flux through carnitine palmitoyltransferase-I (CPT-I), an enzyme located on the outer mitochondrial membrane. Malonyl-CoA (M-CoA) is a naturally occurring inhibitor of CPT-I; therefore, the abundance of M-CoA has long been considered a major regulator of fatty acid oxidation. A recent paper published in the Biochemical Journal by Altamimi et al. ( Biochem. J. (2018) 475 , 959–976) provides evidence for a novel mechanism to produce M-CoA. Specifically, these authors identified carnitine acetyltransferase within the cytosol and further show that flux in the reverse direction forms acetyl-CoA, which is the necessary substrate for the subsequent synthesis of M-CoA. The elegant study design and intriguing data presented by Altamimi et al. provide further insights into the reciprocal regulation of substrate selection within the heart, with implications for fuel utilization and the development of cardiac diseases.

The photosynthetic chloroplast thylakoid membrane of higher plants is a complex three-dimensional structure that is morphologically dynamic on a timescale of just a few minutes. The membrane dynamics are driven by the phosphorylation of light-harvesting complex II (LHCII) by the STN7 kinase, which controls the size of the stacked grana region relative to the unstacked stromal lamellae region. Here, I hypothesise that the functional significance of these membrane dynamics is in controlling the partition of electrons between photosynthetic linear and cyclic electron transfer (LET and CET), which determines the ratio of NADPH/ATP produced. The STN7 kinase responds to the metabolic state of the chloroplast by sensing the stromal redox state. A high NADPH/ATP ratio leads to reduction of thioredoxin f (TRX f ), which reduces a CxxxC motif in the stromal domain of STN7 leading to its inactivation, whereas a low NADPH/ATP ratio leads to oxidation of TRX f and STN7 activation. Phosphorylation of LHCII leads to smaller grana, which favour LET by speeding up diffusion of electron carriers plastoquinone (PQ) and plastocyanin (PC) between the domains. In contrast, dephosphorylation of LHCII leads to larger grana that slow the diffusion of PQ and PC, leaving the PQ pool in the stroma more oxidised, thus enhancing the efficiency of CET. The feedback regulation of electron transfer by the downstream metabolism is crucial to plant fitness, since perturbations in the NADPH/ATP ratio can rapidly lead to the inhibition of photosynthesis and photo-oxidative stress.

Bioenergetic requirements of hematopoietic stem cells and pluripotent stem cells (PSCs) vary with lineage fate, and cellular adaptations rely largely on substrate (glucose/glutamine) availability and mitochondrial function to balance tricarboxylic acid (TCA)-derived anabolic and redox-regulated antioxidant functions. Heme synthesis and degradation converge in a linear pathway that utilizes TCA cycle-derived carbon in cataplerotic reactions of tetrapyrrole biosynthesis, terminated by NAD(P)H-dependent biliverdin reductases (IXα, BLVRA and IXβ, BLVRB) that lead to bilirubin generation and cellular antioxidant functions. We now demonstrate that PSCs with targeted deletion of BLVRB display physiologically defective antioxidant activity and cellular viability, associated with a glutamine-restricted defect in TCA entry that was computationally predicted using gene/metabolite topological network analysis and subsequently validated by bioenergetic and isotopomeric studies. Defective BLVRB-regulated glutamine utilization was accompanied by exaggerated glycolytic accumulation of the rate-limiting hexokinase reaction product glucose-6-phosphate. BLVRB -deficient embryoid body formation (a critical size parameter of early lineage fate potential) demonstrated enhanced sensitivity to the pentose phosphate pathway (PPP) inhibitor 6-aminonicotinamide with no differences in the glycolytic pathway inhibitor 2-deoxyglucose. These collective data place heme catabolism in a crucial pathway of glutamine-regulated bioenergetic metabolism and suggest that early stages of lineage fate potential require glutamine anaplerotic functions and an intact PPP, which are, in part, regulated by BLVRB activity. In principle, BLVRB inhibition represents an alternative strategy for modulating cellular glutamine utilization with consequences for cancer and hematopoietic metabolism.

Chronic metabolic stress leads to cellular dysfunction, characterized by excessive reactive oxygen species, endoplasmic reticulum (ER) stress and inflammation, which has been implicated in the pathogenesis of obesity, type 2 diabetes and cardiovascular disease. The ER is gaining recognition as a key organelle in integrating cellular stress responses. ER homeostasis is tightly regulated by a complex antioxidant system, which includes the seven ER-resident selenoproteins — 15 kDa selenoprotein, type 2 iodothyronine deiodinase and selenoproteins S, N, K, M and T. Here, the findings from biochemical, cell-based and mouse studies investigating the function of ER-resident selenoproteins are reviewed. Human experimental and genetic studies are drawn upon to highlight the relevance of these selenoproteins to the pathogenesis of metabolic disease. ER-resident selenoproteins have discrete roles in the regulation of oxidative, ER and inflammatory stress responses, as well as intracellular calcium homeostasis. To date, only two of these ER-resident selenoproteins, selenoproteins S and N have been implicated in human disease. Nonetheless, the potential of all seven ER-resident selenoproteins to ameliorate metabolic dysfunction warrants further investigation.

Exposure to the toxins methylene cyclopropyl acetic acid (MCPA) and methylene cyclopropyl glycine (MCPG) of unripe ackee and litchi fruit can lead to hypoglycemia and death; however, the molecular mechanisms by which MCPA and MCPG cause hypoglycemia have not been established in vivo . To determine the in vivo mechanisms of action of these toxins, we infused them into conscious rodents and assessed rates of hepatic gluconeogenesis and ketogenesis, hepatic acyl-CoA and hepatic acetyl-CoA content, and hepatocellular energy charge. MCPG suppressed rates of hepatic β-oxidation as reflected by reductions in hepatic ketogenesis, reducing both short- and medium-chain hepatic acyl-CoA concentrations. Hepatic acetyl-CoA content decreased, and hepatic glucose production was inhibited. MCPA also suppressed β-oxidation of short-chain acyl-CoAs, rapidly inhibiting hepatic ketogenesis and hepatic glucose production, depleting hepatic acetyl-CoA content and ATP content, while increasing other short-chain acyl-CoAs. Utilizing a recently developed positional isotopomer NMR tracer analysis method, we demonstrated that MCPA-induced reductions in hepatic acetyl-CoA content were associated with a marked reduction of hepatic pyruvate carboxylase (PC) flux. Taken together, these data reveal the in vivo mechanisms of action of MCPA and MCPG: the hypoglycemia associated with ingestion of these toxins can be ascribed mostly to MCPA- or MCPG-induced reductions in hepatic PC flux due to inhibition of β-oxidation of short-chain acyl-CoAs by MCPA or inhibition of both short- and medium-chain acyl-CoAs by MCPG with resultant reductions in hepatic acetyl-CoA content, with an additional contribution to hypoglycemia through reduced hepatic ATP stores by MCPA.

Obesity represents one of the most complex public health challenges and has recently reached epidemic proportions. Obesity is also considered to be primarily responsible for the rising prevalence of metabolic syndrome, defined as the coexistence in the same individual of several risk factors for atherosclerosis, including dyslipidemia, hypertension and hyperglycemia, as well as for cancer. Additionally, the presence of three of the five risk factors (abdominal obesity, low high-density lipoprotein cholesterol, high triglycerides, high fasting glucose and high blood pressure) characterizes metabolic syndrome, which has serious clinical consequences. The current study was conducted in order to identify metabolic differences in visceral adipose tissue (VAT) collected from obese (body mass index 43–48) human subjects who were diagnosed with metabolic syndrome, obese individuals who were metabolically healthy and nonobese healthy controls. Extensive gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS/MS) analyses were used to obtain the untargeted VAT metabolomic profiles of 481 metabolites belonging to all biochemical pathways. Our results indicated consistent increases in oxidative stress markers from the pathologically obese samples in addition to subtle markers of elevated glucose levels that may be consistent with metabolic syndrome. In the tissue derived from the pathologically obese subjects, there were significantly elevated levels of plasmalogens, which may be increased in response to oxidative changes in addition to changes in glycerolphosphorylcholine, glycerolphosphorylethanolamine glycerolphosphorylserine, ceramides and sphingolipids. These data could be potentially helpful for recognizing new pathways that underlie the metabolic–vascular complications of obesity and may lead to the development of innovative targeted therapies.

The role of carnitine acetyltransferase (CrAT) in regulating cardiac energy metabolism is poorly understood. CrAT modulates mitochondrial acetyl-CoA/CoA (coenzyme A) ratios, thus regulating pyruvate dehydrogenase activity and glucose oxidation. Here, we propose that cardiac CrAT also provides cytosolic acetyl-CoA for the production of malonyl-CoA, a potent inhibitor of fatty acid oxidation. We show that in the murine cardiomyocyte cytosol, reverse CrAT activity (RCrAT, producing acetyl-CoA) is higher compared with the liver, which primarily uses ATP-citrate lyase to produce cytosolic acetyl-CoA for lipogenesis. The heart displayed a lower RCrAT K m for CoA compared with the liver. Furthermore, cytosolic RCrAT accounted for 4.6 ± 0.7% of total activity in heart tissue and 12.7 ± 0.2% in H9C2 cells, while highly purified heart cytosolic fractions showed significant CrAT protein levels. To investigate the relationship between CrAT and acetyl-CoA carboxylase (ACC), the cytosolic enzyme catalyzing malonyl-CoA production from acetyl-CoA, we studied ACC2-knockout mouse hearts which showed decreased CrAT protein levels and activity, associated with increased palmitate oxidation and acetyl-CoA/CoA ratio compared with controls. Conversely, feeding mice a high-fat diet for 10 weeks increased cardiac CrAT protein levels and activity, associated with a reduced acetyl-CoA/CoA ratio and glucose oxidation. These data support the presence of a cytosolic CrAT with a low K m for CoA, favoring the formation of cytosolic acetyl-CoA, providing an additional source to the classical ATP-citrate lyase pathway, and that there is an inverse relation between CrAT and the ratio of acetyl-CoA/CoA as evident in conditions affecting the regulation of cardiac energy metabolism.

The fatty acid 2-hydroxylase (FA2H) is essential for synthesis of 2-hydroxylated fatty acids in myelinating and other cells, and deficiency of this enzyme causes a complicated form of hereditary spastic paraplegia also known as fatty acid hydroxylase-associated neurodegeneration. Despite its important role in sphingolipid metabolism, regulation of FA2H and its interaction with other proteins involved in the same or other metabolic pathways is poorly understood. To identify potential interaction partners of the enzyme, quantitative mass spectrometry using stable isotope labeling of cells was combined with formaldehyde cross-linking and proximity biotinylation, respectively. Besides other enzymes involved in sphingolipid synthesis and intermembrane transfer of ceramide, and putative redox partners of FA2H, progesterone receptor membrane component-1 (PGRMC1) and PGRMC2 were identified as putative interaction partners. These two related heme-binding proteins are known to regulate several cytochrome P450 enzymes. Bimolecular fluorescence complementation experiments confirmed the interaction of FA2H with PGRMC1. Moreover, the PGRMC1 inhibitor AG-205 significantly reduced synthesis of hydroxylated ceramide and glucosylceramide in FA2H-expressing cells. This suggests that PGRMC1 may regulate FA2H activity, possibly through its heme chaperone activity.

The pantothenate (vitamin B 5 ) synthesis pathway in plants is not fully defined because the subcellular site of its ketopantoate → pantoate reduction step is unclear. However, the pathway is known to be split between cytosol, mitochondria, and potentially plastids, and inferred to involve mitochondrial or plastidial transport of ketopantoate or pantoate. No proteins that mediate these transport steps have been identified. Comparative genomic and transcriptomic analyses identified Arabidopsis thaliana BASS1 (At1g78560) and its maize ( Zea mays ) ortholog as candidates for such a transport role. BASS1 proteins belong to the bile acid : sodium symporter family and share similarity with the Salmonella enterica PanS pantoate/ketopantoate transporter and with predicted bacterial transporters whose genes cluster on the chromosome with pantothenate synthesis genes. Furthermore, Arabidopsis BASS1 is co-expressed with genes related to metabolism of coenzyme A, the cofactor derived from pantothenate. Expression of Arabidopsis or maize BASS1 promoted the growth of a S. enterica panB panS mutant strain when pantoate, but not ketopantoate, was supplied, and increased the rate of [ 3 H]pantoate uptake. Subcellular localization of green fluorescent protein fusions in Nicotiana tabacum BY-2 cells demonstrated that Arabidopsis BASS1 is targeted solely to the plastid inner envelope. Two independent Arabidopsis BASS1 knockout mutants accumulated pantoate ∼10-fold in leaves and had smaller seeds. Taken together, these data indicate that BASS1 is a physiologically significant plastidial pantoate transporter and that the pantoate reduction step in pantothenate biosynthesis could be at least partly localized in plastids.

Propionic acidemia is the accumulation of propionate in blood due to dysfunction of propionyl-CoA carboxylase. The condition causes lethargy and striatal degeneration with motor impairment in humans. How propionate exerts its toxic effect is unclear. Here, we show that intravenous administration of propionate causes dose-dependent propionate accumulation in the brain and transient lethargy in mice. Propionate, an inhibitor of histone deacetylase, entered GABAergic neurons, as could be seen from increased neuronal histone H4 acetylation in the striatum and neocortex. Propionate caused an increase in GABA (γ-amino butyric acid) levels in the brain, suggesting inhibition of GABA breakdown. In vitro propionate inhibited GABA transaminase with a K i of ∼1 mmol/l. In isolated nerve endings, propionate caused increased release of GABA to the extracellular fluid. In vivo , propionate reduced cerebral glucose metabolism in both striatum and neocortex. We conclude that propionate-induced inhibition of GABA transaminase causes accumulation of GABA in the brain, leading to increased extracellular GABA concentration, which inhibits neuronal activity and causes lethargy. Propionate-mediated inhibition of neuronal GABA transaminase, an enzyme of the inner mitochondrial membrane, indicates entry of propionate into neuronal mitochondria. However, previous work has shown that neurons are unable to metabolize propionate oxidatively, leading us to conclude that propionyl-CoA synthetase is probably absent from neuronal mitochondria. Propionate-induced inhibition of energy metabolism in GABAergic neurons may render the striatum, in which >90% of the neurons are GABAergic, particularly vulnerable to degeneration in propionic acidemia.

Members of the cytochrome P450 monooxygenase family CYP268 are found across a broad range of Mycobacterium species including the pathogens Mycobacterium avium , M. colombiense , M. kansasii , and M . marinum . CYP268A2, from M. marinum , which is the first member of this family to be studied, was purified and characterised. CYP268A2 was found to bind a variety of substrates with high affinity, including branched and straight chain fatty acids (C10–C12), acetate esters, and aromatic compounds. The enzyme was also found to bind phenylimidazole inhibitors but not larger azoles, such as ketoconazole. The monooxygenase activity of CYP268A2 was efficiently reconstituted using heterologous electron transfer partner proteins. CYP268A2 hydroxylated geranyl acetate and tran s-pseudoionone at a terminal methyl group to yield ( 2E , 6E )-8-hydroxy-3,7-dimethylocta-2,6-dien-1-yl acetate and ( 3E , 5E , 9E )-11-hydroxy-6,10-dimethylundeca-3,5,9-trien-2-one, respectively. The X-ray crystal structure of CYP268A2 was solved to a resolution of 2.0 Å with trans -pseudoionone bound in the active site. The overall structure was similar to that of the related phytanic acid monooxygenase CYP124A1 enzyme from Mycobacterium tuberculosis , which shares 41% sequence identity. The active site is predominantly hydrophobic, but includes the Ser99 and Gln209 residues which form hydrogen bonds with the terminal carbonyl group of the pseudoionone. The structure provided an explanation on why CYP268A2 shows a preference for shorter substrates over the longer chain fatty acids which bind to CYP124A1 and the selective nature of the catalysed monooxygenase activity.

Macrophage foam cells store excess cholesterol as cholesteryl esters, which need to be hydrolyzed for cholesterol efflux. We recently reported that silencing expression of carboxylesterase 1 (CES1) in human THP-1 macrophages [CES1KD (THP-1 cells with CES1 expression knocked down) macrophages] reduced cholesterol uptake and decreased expression of CD36 and scavenger receptor-A in cells loaded with acetylated low-density lipoprotein (acLDL). Here, we report that CES1KD macrophages exhibit reduced transcription of cytochrome P45027A1 ( CYP27A1 ) in nonloaded and acLDL-loaded cells. Moreover, levels of CYP27A1 protein and its enzymatic product, 27-hydroxycholesterol, were markedly reduced in CES1KD macrophages. Transcription of LXRα (liver X receptor α) and ABCA1 (ATP-binding cassette transporter A1) was also decreased in acLDL-loaded CES1KD macrophages, suggesting reduced signaling through PPARγ–CYP27A1–LXRα. Consistent with this, treatment of CES1KD macrophages with agonists for PPARγ, RAR, and/or RAR/RXR partially restored transcription of CYP27A1 and LXRα , and repaired cholesterol influx. Conversely, treatment of control macrophages with antagonists for PPARγ and/or RXR decreased transcription of CYP27A1 and LXRα . Pharmacologic inhibition of CES1 in both wild-type THP-1 cells and primary human macrophages also decreased CYP27A1 transcription. CES1 silencing did not affect transcript levels of PPARγ and RXR in acLDL-loaded macrophages, whereas it did reduce the catabolism of the endocannabinoid 2-arachidonoylglycerol. Finally, the gene expression profile of CES1KD macrophages was similar to that of PPARγ knockdown cells following acLDL exposures, further suggesting a mechanistic link between CES1 and PPARγ. These results are consistent with a model in which abrogation of CES1 function attenuates the CYP27A1–LXRα–ABCA1 signaling axis by depleting endogenous ligands for the nuclear receptors PPARγ, RAR, and/or RXR that regulate cholesterol homeostasis.