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Diabetic cardiomyopathy describes heart disease in patients with diabetes who have no other cardiac conditions but have a higher risk of developing heart failure. Specific therapies to treat the diabetic heart are limited. A key mechanism involved in the progression of diabetic cardiomyopathy is dysregulation of cardiac energy metabolism. The aim of this study was to determine if increasing the expression of medium-chain acyl-coenzyme A dehydrogenase (MCAD; encoded by Acadm), a key regulator of fatty acid oxidation, could improve the function of the diabetic heart. Male mice were administered streptozotocin to induce diabetes, which led to diastolic dysfunction 8 weeks post-injection. Mice then received cardiac-selective adeno-associated viral vectors encoding MCAD (rAAV6:MCAD) or control AAV and were followed for 8 weeks. In the non-diabetic heart, rAAV6:MCAD increased MCAD expression (mRNA and protein) and increased Acadl and Acadvl, but an increase in MCAD enzyme activity was not detectable. rAAV6:MCAD delivery in the diabetic heart increased MCAD mRNA expression but did not significantly increase protein, activity, or improve diabetes-induced cardiac pathology or molecular metabolic and lipid markers. The uptake of AAV viral vectors was reduced in the diabetic versus non-diabetic heart, which may have implications for the translation of AAV therapies into the clinic. Key messages: The effects of increasing MCAD in the diabetic heart are unknown. Delivery of rAAV6:MCAD increased MCAD mRNA and protein, but not enzyme activity, in the non-diabetic heart. Independent of MCAD enzyme activity, rAAV6:MCAD increased Acadl and Acadvl in the non-diabetic heart. Increasing MCAD cardiac gene expression alone was not sufficient to protect against diabetes-induced cardiac pathology. AAV transduction efficiency was reduced in the diabetic heart, which has clinical implications. [ABSTRACT FROM AUTHOR]

Significance: Glioblastoma is an aggressive and devastating brain tumor characterized by a dismal prognosis and resistance to therapeutic intervention. To support catabolic processes critical for unabated cellular growth and defend against harmful reactive oxygen species, glioblastoma tumors upregulate the expression of wild-type isocitrate dehydrogenases (IDHs). IDH enzymes catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), NAD(P)H, and CO2. On molecular levels, IDHs epigenetically control gene expression through effects on α-KG-dependent dioxygenases, maintain redox balance, and promote anaplerosis by providing cells with NADPH and precursor substrates for macromolecular synthesis. Recent Advances: While gain-of-function mutations in IDH1 and IDH2 represent one of the most comprehensively studied mechanisms of IDH pathogenic effects, recent studies identified wild-type IDHs as critical regulators of normal organ physiology and, when transcriptionally induced or down regulated, as contributing to glioblastoma progression. Critical Issues: Here, we will discuss molecular mechanisms of how wild-type IDHs control glioma pathogenesis, including the regulation of oxidative stress and de novo lipid biosynthesis, and provide an overview of current and future research directives that aim to fully characterize wild-type IDH-driven metabolic reprogramming and its contribution to the pathogenesis of glioblastoma. Future Directions: Future studies are required to further dissect mechanisms of metabolic and epigenomic reprogramming in tumors and the tumor microenvironment, and to develop pharmacological approaches to inhibit wild-type IDH function. Antioxid. Redox Signal. 39, 923–941. [ABSTRACT FROM AUTHOR]

A 3-year-old, male neutered Cavalier King Charles Spaniel (CKCS) presented with complex focal seizures and prolonged lethargy. The aim of the study was to investigate the clinical signs, metabolic changes and underlying genetic defect. Blood and urine organic acid analysis revealed increased medium-chain fatty acids and together with the clinical findings suggested a diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. We sequenced the genome of the affected dog and compared the data to 923 control genomes of different dog breeds. The ACADM gene encoding MCAD was considered the top functional candidate gene. The genetic analysis revealed a single homozygous private protein-changing variant in ACADM in the affected dog. This variant, XM_038541645.1:c.444_445delinsGTTAATTCTCAATATTGTCTAAGAATTATG, introduces a premature stop codon and is predicted to result in truncation of ~63% of the wild type MCAD open reading frame, XP_038397573.1:p.(Thr150Ilefs*6). Targeted genotyping of the variant in 162 additional CKCS revealed a variant allele frequency of 23.5% and twelve additional homozygous mutant dogs. The acylcarnitine C8/C12 ratio was elevated ~43.3 fold in homozygous mutant dogs as compared to homozygous wild type dogs. Based on available clinical and biochemical data together with current knowledge in humans, we propose the ACADM frameshift variant as causative variant for the MCAD deficiency with likely contribution to the neurological phenotype in the index case. Testing the CKCS breeding population for the identified ACADM variant is recommended to prevent the unintentional breeding of dogs with MCAD deficiency. Further prospective studies are warranted to assess the clinical consequences of this enzyme defect. [ABSTRACT FROM AUTHOR]

Lipoic acid is an organosulfur cofactor essential for several key enzyme complexes in oxidative and one-carbon metabolism. It is covalently bound to the lipoyl domain of the E2 subunit in some 2-oxoacid dehydrogenase complexes and the H-protein in the glycine cleavage system. Lipoate-protein ligase (Lpl) is involved in the salvage of exogenous lipoate and attaches free lipoate to the E2 subunit or the H-protein in an ATP-dependent manner. In the hyperthermophilic archaeon Thermococcus kodakarensis, TK1234 and TK1908 are predicted to encode the N- and C-terminal regions of Lpl, respectively. TK1908 and TK1234 recombinant proteins form a heterodimer and together displayed significant ligase activity toward octanoate in addition to lipoate when a chemically synthesized octapeptide was used as the acceptor. The proteins also displayed activity toward other fatty acids, indicating broad fatty acid specificity. On the other hand, lipoyl synthase from T. kodakarensis only recognized octanoyl-peptide as a substrate. Examination of individual proteins indicated that the TK1908 protein alone was able to catalyze the ligase reaction although with a much lower activity. Gene disruption of TK1908 led to lipoate/serine auxotrophy, whereas TK1234 gene deletion did not. Acyl carrier protein homologs are not found on the archaeal genomes, and the TK1908/TK1234 protein complex did not utilize octanoyl-CoA, raising the possibility that the substrate of the ligase reaction is octanoic acid itself. Although Lpl has been considered as an enzyme involved in lipoate salvage, the results imply that in T. kodakarensis, the TK1908 and TK1234 proteins function in de novo lipoyl-protein biosynthesis. [ABSTRACT FROM AUTHOR]

Background: Hypoxia signaling, especially the hypoxia inducible factor (HIF) pathway, is a major player in clear cell renal cell carcinoma (ccRCC), which is characterized by disorders in lipid and glycogen metabolism. However, the interaction between hypoxia and lipid metabolism in ccRCC progression is still poorly understood. Methods: We used bioinformatic analysis and discovered that glycerol-3-phosphate dehydrogenase 1 (GPD1) may play a key role in hypoxia and lipid metabolism pathways in ccRCC. Tissue microarray, IHC staining, and survival analysis were performed to evaluate clinical function. In vitro and in vivo assays showed the biological effects of GPD1 in ccRCC progression. Results: We found that the expression of GPD1 was downregulated in ccRCC tissues, and overexpression of GPD1 inhibited the progression of ccRCC both in vivo and in vitro. Furthermore, we demonstrated that hypoxia inducible factor-1α (HIF1α) directly regulates GPD1 at the transcriptional level, which leads to the inhibition of mitochondrial function and lipid metabolism. Additionally, GPD1 was shown to inhibit prolyl hydroxylase 3 (PHD3), which blocks prolyl-hydroxylation of HIF1α and subsequent proteasomal degradation, and thus reinforces the inhibition of mitochondrial function and phosphorylation of AMPK via suppressing glycerol-3-phosphate dehydrogenase 2 (GPD2). Conclusions: This study not only demonstrated that HIF1α-GPD1 forms a positive feedforward loop inhibiting mitochondrial function and lipid metabolism in ccRCC, but also discovered a new mechanism for the molecular basis of HIF1α to inhibit tumor activity, thus providing novel insights into hypoxia-lipid-mediated ccRCC therapy. [ABSTRACT FROM AUTHOR]

The aim of the study was to examine the features of phenotype and the levels of NAD(P)-dependent dehydrogenases activity of blood neutrophils in the prognosis of abdominal sepsis development in patients with widespread purulent peritonitis (WPP). 50 patients with WPP of community and hospital origin in the pre-operative period were examined. From the 5th to the 10th day of the postoperative period 35 patients (70%) had developed abdominal sepsis, 15 patients (30%) had absence of complications. As controls 67 respect healthy people were examined. The research blood neutrophils phenotype was performed by f low cytometry using a direct immunofluorescence whole peripheral blood. The levels of surface receptor expression was assessed by the mean fluorescence intensity. The NAD(P)-dependent dehydrogenases activity in the blood neutrophils studied using bioluminescence method. It was established that the inflammatory reaction in patients with WPP is characterized by neutrophilia and changes in the phenotype of blood neutrophils. The markers of the subsequent development of sepsis in WPP are less pronounced (in comparison with the indices of uncomplicated patients), an increase in the number of neutrophils, a decrease in the HLA-DR + -cell count against the background of a high level of neutrophils expressing a high affinity receptor for IgG. The patients without subsequent complications had the number of neutrophils with CD23 receptor expression is increased. Metabolism of neutrophils in patients with WPP is characterized by a decrease in the intensity of plastic processes due to low activity of glucose-6-phosphate dehydrogenase and an imbalance in the activity of the enzymes of the mitochondrial compartment. A feature of the neutrophil metabolism in patients with WPP without subsequent development of sepsis is a high activity of anaerobic lactate dehydrogenase reaction and a decrease in the activity of NADP-dependent decarboxylating malate dehydrogenase. The patients with WPP with subsequent development of sepsis had a high level of NAD-dependent out flow of substrates from the tricarboxylic acid cycle on the amino acid exchange reaction via glutamate dehydrogenase which can affect the activity of aerobic respiration of blood neutrophils. The established differences in the phenotype and activity of enzymes in the blood neutrophils in patients with WPP depending on the subsequent development of sepsis determine the possibility of creating a method for predicting the development of complications and developing immunoactive therapy in the postoperative period of WPP.

Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways: [ABSTRACT FROM AUTHOR]

Despite physiological importance of aldonic sugar acids for living organisms, little is known about metabolic pathways of these compounds. Here, we investigated the functional diversity of homologs of L-threonic acid dehydrogenase (ThrDH; UniProt ID: Q0KBC7), an enzyme composed of two NAD-binding domains (PF14833 and PF03446). Ten ThrDH homologs with different genomic context were studied; seven new enzymatic activities were identified, such as (R)-pantoate dehydrogenase, L-altronic acid dehydrogenase, 6-deoxy-L-talonate dehydrogenase, L-idonic acid dehydrogenase, D-xylonic acid dehydrogenase, D-gluconic acid dehydrogenase, and 2-hydroxy-3-oxopantoate reductase activities. Two associated metabolic pathways were identified: L-idonic acid dehydrogenase was found to be involved in the degradation of L-idonic acid through oxidation/decarboxylation in Agrobacterium radiobacter K84, while 2-hydroxy-3-oxopantoate reductase was found to participate in D-glucarate catabolism through dehydration/cleavage in Ralstonia metallidurans CH34. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa can metabolize acyclic monoterpenoids (such as citronellol and geraniol) as the only carbon and energy sources. A total of seven proteins (AtuA, AtuB, AtuCF, AtuD, AtuE, AtuG, AtuH) have been identified in Pseudomonas aeruginosa as participating in the acyclic terpene utilization pathway. AtuB is a dehydrogenase enzyme responsible for citronellol and geraniol catabolism in the acyclic terpene utilization (Atu) pathway, although its structure and function have not been characterized to date. Here we report the crystal structure of AtuB from Pseudomonas aeruginosa PAO1 (PaAtuB) to 1.8 Å resolution. PaAtuB crystallizes in the space group F222 with a single monomer in the asymmetric unit. Analytical ultracentrifugation data shows that PaAtuB forms a stable tetramer in solution, which is consistent with the structure. Structural analysis confirms that AtuB belongs to the short-chain dehydrogenase/reductase (SDR) family. AtuB is predicted to bind NADP(H) from the crystal structure, which is confirmed by MicroScale Thermophoresis analysis that shows PaAtuB binds NADP(H) with a Kd value of 258 μM. This work provides a starting point to explore potential biotechnology and pharmaceutical applications of AtuB. • A crystal structure of P. aeruginosa dehydrogenase AtuB was determined. • P. aeruginosa AtuB was confirmed to be a member of the short-chain dehydrogenase/reductase (SDR) family. • P. aeruginosa AtuB forms a stable tetramer in solution. • P. aeruginosa AtuB is confirmed to bind NADP(H). • The AtuB structure should provide a starting point to explore industrial applications in citronellol and geraniol catabolism. [ABSTRACT FROM AUTHOR]

Monoamine neurotransmitters play essential roles in the regulation of arousal and sleep. Impaired metabolism of monoamine neurotransmitters could result in the accumulation of neurotoxic aldehyde metabolites and, hence, neuronal degeneration. Aldehyde dehydrogenases play an important role in the metabolism of the neurotoxic aldehyde metabolites, including the aldehyde metabolites of dopamine, serotonin, and noradrenaline. Deficient aldehyde dehydrogenase 2 (ALDH2) has been suggested to result in the accumulation of these biogenic aldehydes. An ALDH2 single nucleotide polymorphism (SNP), rs671 (A), results in significantly reduced ALDH2 enzyme activity. A total of 83 Parkinson's disease (PD) patients were recruited in this study. In addition to the genotypes of rs671, the patients were assessed with the PD sleep scale-2nd version (PDSS-2) and the Epworth sleepiness scale (ESS) for symptoms of daytime and nocturnal sleep disturbances. The patients carrying rs671 (A) had more frequent dozing while lying down to rest in the afternoon (ESS item5) (F = 7.308, p = 0.008) than the rs671 (GG) patients. The patients with rs671 (A) reported a trend toward more frequent difficulty staying asleep than the patients with rs671 (GG). (F = 3.278, p = 0.074). The results indicate that patients carrying allele rs671 (A) are more likely to experience impairment in the regulation of arousal and sleep. The results also support the hypothesis that the accumulation of neurotoxic monoamine neurotransmitter aldehyde metabolites secondary to reduced ALDH2 enzyme activity may cause more severe monoaminergic neuronal loss and, hence, more severe symptoms in the regulation of wakefulness and sleep. [ABSTRACT FROM AUTHOR]

Mycobacterium smegmatis and several other mycobacteria are able to utilize methanol as the sole source of carbon and energy. We recently showed that N,N-dimethyl-p-nitrosoaniline (NDMA)-dependent methanol dehydrogenase (Mno) is essential for the growth of M. smegmatis on methanol. Although Mno from this bacterium shares high homology with other known methanol dehydrogenases, methanol metabolism in M. smegmatis differs significantly from that of other described methylotrophs. In this study, we dissect the regulatory mechanism involved in the methylotrophic metabolism in M. smegmatis. We identify a two-component system (TCS), mnoSR, that is involved in the regulation of mno expression. We show that the MnoSR TCS is comprised of a sensor kinase (MnoS) and a response regulator (MnoR). Our results demonstrate that MnoS undergoes autophosphorylation and is able to transfer its phosphate to MnoR by means of phosphotransferase activity. Furthermore, MnoR shows specific binding to the putative mno promoter region in vitro, thus suggesting its role in the regulation of mno expression. Additionally, we find that the MnoSR system is involved in the regulation of MSMEG_6239, which codes for a putative 1,3-propanediol dehydrogenase. We further show that M. smegmatis lacking mnoSR is unable to utilize methanol and 1,3-propanediol as the sole carbon source, which confirms the role of MnoSR in the regulation of alcohol metabolism. Our data, thus, suggest that the regulation of mno expression in M. smegmatis provides new insight into the regulation of methanol metabolism, which furthers our understanding of methylotrophy in mycobacteria. [ABSTRACT FROM AUTHOR]

Abstract A Pseudomonas sp. DNE-S1 (GenBank accession number MF803832), able to degrade DEP in a wide range of acid-base conditions, was isolated from landfill soil. The growth kinetics of DNE-S1 on DEP followed the inhibition model. Fe3+ could promote the degradation ability of DNE-S1 to DEP probably by over-expression of the gene phthalate dihydrodiol dehydrogenase (ophB) and phthalate dioxygenase ferredoxin reductase (ophA4). The degradation rate of DEP (500 mg L−1 at 12 h) increased by 14.5% in the presence of Fe3+. Cu2+, Zn2+, and Mn2+ showed an inhibiting effect on the degradation performance of the strain and could alter the cellular morphology, surface area and volume of DNE-S1. Three degradation intermediates, namely ethyl methyl phthalate (EMP), dimethyl phthalate (DMP), and phthalic acid (PA), were detected in the biodegradation of DEP, and the biochemical pathway of DEP degradation was proposed. This study provides new information on the biochemical pathways and the responsible genes involved in DEP degradation. Highlights • Pseudomonas sp. DNE-S1 was able to degrade DEP in a wide range of acid-base conditions. • Two phthalate degrading genes ophB and ophA4 were identified in this bacterium. • Cellular morphology, surface area and volume were altered upon exposure to trace metal. • Fe3+ could promote the DEP degradation of DNE-S1. [ABSTRACT FROM AUTHOR]

Extensive use of nanoparticles at commercial level inevitably enhances their release into the environment and effects terrestrial as well as aquatic organisms. The entry of nanoparticles in soil ecosystem causes toxicity in animals living there in. So an experiment was conducted to study the toxicological effects of zinc oxide nanoparticles (ZnO NPs) on some biochemical constituents of the earthworm, Eisenia fetida. The individuals of the earthworm were exposed to different concentrations of ZnO NPs for two weeks. The sublethal concentrations of ZnO NPs significantly declined the DNA, RNA and protein contents. Maximum reduction was observed in protein, while minimum decline was in RNA/DNA ratio. Similarly, the treatment of ZnO NPs decreased the carbohydrate and lipid contents in E. fetida. The decrease in carbohydrate content was more as compared to decline in lipid. The ZnO NPs significantly declined the activity of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH). The reduction in activity was more in MDH as compared to LDH indicating greater impact on aerobic capacity of the earthworm. Nanoparticle associated decrease in dehydrogenase activity reflected inhibition in metabolic functions. The effects of ZnO NPs on biochemical constituents of earthworm were concentration-dependent. It suggested that the use of nanoparticles should be minimized as far as possible to protect metabolism and health of earthworms. [ABSTRACT FROM AUTHOR]

Non-methanotrophic bacteria such as methylotrophs often coexist with methane-oxidizing bacteria (methanotrophs) by cross-feeding on methane-derived carbon. Methanol has long been considered a major compound that mediates cross-feeding of methane-derived carbon. Despite the potential importance of cross-feeding in the global carbon cycle, only a few studies have actually explored metabolic responses of a bacteria when cross-feeding on a methanotroph. Recently, we isolated a novel facultative methylotroph, Methyloceanibacter caenitepidi Gela4, which grows syntrophically with the methanotroph, Methylocaldum marinum S8. To assess the potential metabolic pathways in M. caenitepidi Gela4 co-cultured with M. marinum S8, we conducted genomic analyses of the two strains, as well as RNA-Seq and chemical analyses of M. caenitepidi Gela4, both in pure culture with methanol and in co-culture with methanotrophs. Genes involved in the serine pathway were downregulated in M. caenitepidi Gela4 under co-culture conditions, and methanol was below the detection limit (< 310 nM) in both pure culture of M. marinum S8 and co-culture. In contrast, genes involved in the tricarboxylic acid cycle, as well as acetyl-CoA synthetase, were upregulated in M. caenitepidi Gela4 under co-culture conditions. Notably, a pure culture of M. marinum S8 produced acetate (< 16 μM) during growth. These results suggested that an organic compound other than methanol, possibly acetate, might be the major carbon source for M. caenitepidi Gela4 cross-fed by M. marinum S8. Co-culture of M. caenitepidi Gela4 and M. marinum S8 may represent a model system to further study methanol-independent cross-feeding from methanotrophs to non-methanotrophic bacteria. [ABSTRACT FROM AUTHOR]

Aldehydes are carbonyl compounds found ubiquitously in the environment, derived from both natural and anthropogenic sources. As the aldehydes are reactive species, therefore, they are generally toxic to the body. To reduce the toxicity and pathogenesis related to aldehydes, the human body contains several aldehyde metabolizing enzyme systems including aldehyde oxidases, cytochrome P450 enzymes, aldo-ketoreductases, alcohol dehydrogenases, short-chain dehydrogenases/reductases and aldehyde dehydrogenases (ALDHs). These enzyme systems maintain a low level of aldehydes in the body by catalytically converting them into less-harmful and easily excreted products. The human ALDH (hALDH) superfamily consists of 20 functional ALDH genes identified so far at distinct chromosomal locations, expressing 20 ALDH proteins, which belong to 11 different ALDH families. They are involved in the NAD(P)+-dependent oxidation of a wide range of exogenous and endogenous aldehydes to their corresponding carboxylic acids. The hALDHs are present in all sub-cellular locations and have a wide tissue distribution. This review gives an account of aldehydes; their source, toxicity and metabolism, different aldehyde metabolizing enzymes with special emphasis on ALDHs including their biochemical, physiological and pathophysiological roles in the body. [ABSTRACT FROM AUTHOR]

Highlights • Furfural severely inhibited the butyrate productivity of C. tyrobutyricum. • Heterologous expression of sdr increased furfural tolerance of C. tyrobutyricum. • Coexpression of sdr and groESL enhanced the utilization of corncob acid hydrolysate. • 32.8 g/L butyrate at a productivity of 0.29 g/L·h and yield of 0.40 g/g was obtained. Abstract Resistance to furan derivatives and phenolic compounds plays an important role in the use of lignocellulosic biomass for biological production of chemicals and fuels. This study confirmed that expression of short-chain dehydrogenase/reductase (SDR) from Clostridium beijerinckii NCIMB 8052 significantly improved the tolerance of C. tyrobutyricum to furfural due to the enhanced activity for furfural reduction. And on this basis, co-expression of SDR and heat shock chaperones GroESL could simultaneously enhance the tolerance of C. tyrobutyricum to furan derivatives and phenolic compounds, which were the main inhibitors presented in dilute-acid lignocellulosic hydrolysates. Consequently, the recombinant strain ATCC 25755/ sdr + groESL exhibited good performance in butyric acid production with corncob acid hydrolysate as the substrate. Batch fermentation in bioreactor showed that the butyrate produced by ATCC 25755/ sdr + groESL was 32.8 g/L, increased by 28.1% as compared with the wild-type strain. Meanwhile, the butyrate productivity increased from 0.19 g/L·h to 0.29 g/L·h. [ABSTRACT FROM AUTHOR]

Background: 3‐ketosteroid‐Δ1‐dehydrogenase, a key enzyme involved in microbial catabolism of sterols, catalyzes the 1, 2‐desaturation of steroidal substrates using FAD as a cofactor. Recombinant 3‐ketosteroid‐Δ1‐dehydrogenase from Arthrobacter simplex (KsdD4) was expressed in Escherichia coli BL21 (DE3). Results: KsdD4 exhibited optimal activity at pH 7.0 and 35°C. KsdD4 had the highest activity toward 4‐androstene‐3,17‐dione (AD) and a moderate activity toward cortisone acetate and hydrocortisone. Structure‐based homology modeling revealed that Y118, Y355, Y528 and G532 are located in the active site, thus the most likely catalytic residues. The substrate‐binding cavity was composed of hydrophobic residues with small side chains, including A49, V328, A390 and A531, which facilitate the recognition of steroidal substrates with large C17 side‐chains. E332 forms a unique hydrogen bond with substrates. Recombinant E. coli resting cells expressing KsdD4 showed high catalytic activity in the presence of various organic solvents. A fed‐batch bioconversion using resting cells resulted in efficient production of 2.66 g L−1 androst‐1,4‐diene‐3,17‐dione (ADD). Conclusion: Biochemical data and structural analysis greatly advanced our understanding of the KsdD family enzymes, and the fed‐batch strategy improved the bioconversion of steroids. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Abstract Isobutyryl-CoA dehydrogenase deficiency (IBDHD) is a rare autosomal recessive metabolic disorder related to valine catabolism and results from variants in ACAD8. Here, we present the clinical, biochemical, and genotypes of seven patients with IBDHD in China for the first time. Five patients remained asymptomatic during follow-up, whereas one juvenile had speech delay and one newborn exhibited clinical symptoms. All patients showed remarkably increased concentrations of C4-aclycarnitine with elevated C4/C2 and C4/C3 ratios. In urine organic acid tests, only one patient presented with an increased concentration of isobutyrylglycine excretion. Genetic testing was performed to detect the causative variants. Five previously unreported variants, c.235C > G, c.286G > A, c.444G > T c.1092 + 1G > A, and c.1176G > T, and one known variant, c.1000C > T, in ACAD8 were identified. These previously unreported variants in ACAD8 were predicted to be disease-causing and the c.1092 + 1G > A variant was confirmed to cause skipping of exon 9 by reverse transcription PCR. The most common variant was c.286G > A, which showed an allelic frequency of 50% (7/14), and thus may be a prevalent variant among Chinese patients. Our results broaden the mutational spectrum of ACAD8 and improve the understanding of the clinical phenotype of IBDHD. Highlights • This is the first report of clinical, biochemical, and genetic study on isobutyryl-CoA dehydrogenase deficiency in China. • Five previously unreported variants in ACAD8 gene were identified. • The previously unreported splice variant was confirmed to cause skipping of exon 9 by RT-PCR. • A prevalent variant c.286G > A was identified among Chinese population. [ABSTRACT FROM AUTHOR]

High-level behaviour of metabolic systems results from the properties of, and interactions between, numerous molecular components. Reaching a complete understanding of metabolic behaviour based on the system’s components is therefore a difficult task. This problem can be tackled by constructing and subsequently analysing kinetic models of metabolic pathways since such models aim to capture all the relevant properties of the system components and their interactions. Symbolic control analysis is a framework for analysing pathway models in order to reach a mechanistic understanding of their behaviour. By providing algebraic expressions for the sensitivities of system properties, such as metabolic flux or steady-state concentrations, in terms of the properties of individual reactions it allows one to trace the high level behaviour back to these low level components. Here we apply this method to a model of pyruvate branch metabolism in Lactococcus lactis in order to explain a previously observed negative flux response towards an increase in substrate concentration. With this method we are able to show, first, that the sensitivity of flux towards changes in reaction rates (represented by flux control coefficients) is determined by the individual metabolic branches of the pathway, and second, how the sensitivities of individual reaction rates towards their substrates (represented by elasticity coefficients) contribute to this flux control. We also quantify the contributions of enzyme binding and mass-action to enzyme elasticity separately, which allows for an even finer-grained understanding of flux control. These analytical tools allow us to analyse the control properties of a metabolic model and to arrive at a mechanistic understanding of the quantitative contributions of each of the enzymes to this control. Our analysis provides an example of the descriptive power of the general principles of symbolic control analysis. [ABSTRACT FROM AUTHOR]

The important role of mitochondria in cancer biology is gaining momentum. With their regulation of cell survival, metabolism, basic cell building blocks, and immunity, among other functions, mitochondria affect not only cancer progression but also the response and resistance to current treatments. Calcium ions are constantly shuttled in and out of mitochondria; thus, playing an important role in the regulation of various cellular processes. The mitochondrial calcium uniporter (MCU) channel and its associated regulators transport calcium across the inner mitochondrial membrane to the mitochondrial matrix. Due to this central role and the capacity to affect cell behavior and fate, the MCU complex is being investigated in different cancers and cancer-related conditions. Here, we review current knowledge on the role of the MCU complex in multiple cancer types and models; we also provide a perspective for future research and clinical considerations. [ABSTRACT FROM AUTHOR]

The biological effects of physiological stimuli of adrenocortical glomerulosa cells are predominantly mediated by the Ca2+ and the cAMP signal transduction pathways. The complex interplay between these signalling systems fine-tunes aldosterone secretion. In addition to the well-known cytosolic interactions, a novel intramitochondrial Ca2+-cAMP interplay has been recently recognised. The cytosolic Ca2+ signal is rapidly transferred into the mitochondrial matrix where it activates Ca2+-sensitive dehydrogenases, thus enhancing the formation of NADPH, a cofactor of steroid synthesis. Quite a few cell types, including H295R adrenocortical cells, express the soluble adenylyl cyclase within the mitochondria and the elevation of mitochondrial [Ca2+] activates the enzyme, thus resulting in the Ca2+-dependent formation of cAMP within the mitochondrial matrix. On the other hand, mitochondrial cAMP (mt-cAMP) potentiates the transfer of cytosolic Ca2+ into the mitochondrial matrix. This cAMP-mediated positive feedback control of mitochondrial Ca2+ uptake may facilitate the rapid hormonal response to emergency situations since knockdown of soluble adenylyl cyclase attenuates aldosterone production whereas overexpression of the enzyme facilitates steroidogenesis in vitro. Moreover, the mitochondrial Ca2+-mt-cAMP-Ca2+ uptake feedback loop is not a unique feature of adrenocortical cells; a similar signalling system has been described in HeLa cells as well. [ABSTRACT FROM AUTHOR]

Maple syrup urine disease (MSUD) is a metabolic disorder caused by mutations in three of the branched-chain α-keto acid dehydrogenase complex (BCKDC) genes. Classical MSUD symptom can be observed immediately after birth and include ketoacidosis, irritability, lethargy, and coma, which can lead to death or irreversible neurodevelopmental delay in survivors. The molecular diagnosis of MSUD can be time-consuming and difficult to establish using conventional Sanger sequencing because it could be due to pathogenic variants of any of the BCKDC genes. Next-generation sequencing-based methodologies have revolutionized the molecular diagnosis of inborn errors in metabolism and offer a superior approach for genotyping these patients. Here, we report an MSUD case whose molecular diagnosis was performed by clinical exome sequencing (CES), and the possible structural pathogenic effect of a novel E1α subunit pathogenic variant was analyzed using in silico analysis of α and β subunit crystallographic structure. Molecular analysis revealed a new homozygous non-sense c.1267C>T or p.Gln423Ter variant of BCKDHA . The novel BCKDHA variant is considered pathogenic because it caused a premature stop codon that probably led to the loss of the last 22 amino acid residues of the E1α subunit C-terminal end. In silico analysis of this region showed that it is in contact with several residues of the E1β subunit mainly through polar contacts, hydrogen bonds, and hydrophobic interactions. CES strategy could benefit the patients and families by offering precise and prompt diagnosis and better genetic counseling. [ABSTRACT FROM AUTHOR]

Carboxydothermus species are some of the most studied thermophilic carboxydotrophs. Their varied carboxydotrophic growth properties suggest distinct strategies for energy conservation via carbon monoxide (CO) metabolism. In this study, we used comparative genome analysis of the genus Carboxydothermus to show variations in the CO dehydrogenase-energy-converting hydrogenase gene cluster, which is responsible for CO metabolism with H2 production (hydrogenogenic CO metabolism). Indeed, the ability or inability to produce H2 with CO oxidation is explained by the presence or absence of this gene cluster in Carboxydothermus hydrogenoformans, Carboxydothermus islandicus, and Carboxydothermus ferrireducens. Interestingly, despite its hydrogenogenic CO metabolism, Carboxydothermus pertinax lacks the Ni-CO dehydrogenase catalytic subunit (CooS-I) and its transcriptional regulator-encoding genes in this gene cluster, probably due to inversion. Transcriptional analysis in C. pertinax showed that the Ni-CO dehydrogenase gene (cooS-II) and distantly encoded energy-converting-hydrogenase-related genes were remarkably upregulated with 100% CO. In addition, when thiosulfate was available as a terminal electron acceptor in 100% CO, the maximum cell density and maximum specific growth rate of C. pertinax were 3.1-fold and 1.5-fold higher, respectively, than when thiosulfate was absent. The amount of H2 produced was only 62% of the amount of CO consumed, less than expected according to hydrogenogenic CO oxidation (CO + H2O → CO2 + H2). Accordingly, C. pertinax would couple CO oxidation by Ni-CO dehydrogenase II with simultaneous reduction of not only H2O but also thiosulfate when grown in 100% CO. IMPORTANCE Anaerobic hydrogenogenic carboxydotrophs are thought to fill a vital niche by scavenging potentially toxic CO and producing H2 as an available energy source for thermophilic microbes. This hydrogenogenic carboxydotrophy relies on a Ni-CO dehydrogenase-energy-converting hydrogenase gene cluster. This feature is thought to be common to these organisms. However, the hydrogenogenic carboxydotroph Carboxydothermus pertinax lacks the gene for the Ni-CO dehydrogenase catalytic subunit encoded in the gene cluster. Here, we performed a comparative genome analysis of the genus Carboxydothermus, a transcriptional analysis, and a cultivation study in 100% CO to prove the hydrogenogenic CO metabolism. Results revealed that C. pertinax could couple Ni-CO dehydrogenase II alternatively to the distal energy-converting hydrogenase. Furthermore, C. pertinax represents an example of the functioning of Ni-CO dehydrogenase that does not always correspond to its genomic context, owing to the versatility of CO metabolism and the low redox potential of CO. [ABSTRACT FROM AUTHOR]

The kynurenine pathway is the primary route for L-tryptophan degradation in mammals. Intermediates and side products of this pathway are involved in immune response and neurodegenerative diseases. This makes the study of enzymes, especially those from mammalian sources, of the kynurenine pathway worthwhile. Recent studies on a bacterial version of an enzyme of this pathway, 2-aminomuconate semialdehyde (2-AMS) dehydrogenase (AMSDH), have provided a detailed understanding of the catalytic mechanism and identified residues conserved for muconate semialdehyde recognition and activation. Findings from the bacterial enzyme have prompted the reconsideration of the function of a previously identified human aldehyde dehydrogenase, ALDH8A1 (or ALDH12), which was annotated as a retinal dehydrogenase based on its ability to preferentially oxidize 9-cis-retinal over trans-retinal. Here, we provide compelling bioinformatics and experimental evidence that human ALDH8A1 should be reassigned to the missing 2-AMS dehydrogenase of the kynurenine metabolic pathway. For the first time, the product of the semialdehyde oxidation by AMSDH is also revealed by NMR and high-resolution MS. We found that ALDH8A1 catalyzes the NAD+-dependent oxidation of 2-AMS with a catalytic efficiency equivalent to that of AMSDH from the bacterium Pseudomonas fluorescens. Substitution of active-site residues required for substrate recognition, binding, and isomerization in the bacterial enzyme resulted in human ALDH8A1 variants with 160-fold increased Km or no detectable activity. In conclusion, this molecular study establishes an additional enzymatic step in an important human pathway for tryptophan catabolism. [ABSTRACT FROM AUTHOR]

De novo biosynthesis of lipids is essential for Trypanosoma brucei, a protist responsible for the sleeping sickness. Here, we demonstrate that the ketogenic carbon sources, threonine, acetate and glucose, are precursors for both fatty acid and sterol synthesis, while leucine only contributes to sterol production in the tsetse fly midgut stage of the parasite. Degradation of these carbon sources into lipids was investigated using a combination of reverse genetics and analysis of radio-labelled precursors incorporation into lipids. For instance, (i) deletion of the gene encoding isovaleryl-CoA dehydrogenase, involved in the leucine degradation pathway, abolished leucine incorporation into sterols, and (ii) RNAi-mediated down-regulation of the SCP2-thiolase gene expression abolished incorporation of the three ketogenic carbon sources into sterols. The SCP2-thiolase is part of a unidirectional two-step bridge between the fatty acid precursor, acetyl-CoA, and the precursor of the mevalonate pathway leading to sterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA. Metabolic flux through this bridge is increased either in the isovaleryl-CoA dehydrogenase null mutant or when the degradation of the ketogenic carbon sources is affected. We also observed a preference for fatty acids synthesis from ketogenic carbon sources, since blocking acetyl-CoA production from both glucose and threonine abolished acetate incorporation into sterols, while incorporation of acetate into fatty acids was increased. Interestingly, the growth of the isovaleryl-CoA dehydrogenase null mutant, but not that of the parental cells, is interrupted in the absence of ketogenic carbon sources, including lipids, which demonstrates the essential role of the mevalonate pathway. We concluded that procyclic trypanosomes have a strong preference for fatty acid versus sterol biosynthesis from ketogenic carbon sources, and as a consequence, that leucine is likely to be the main source, if not the only one, used by trypanosomes in the infected insect vector digestive tract to feed the mevalonate pathway. [ABSTRACT FROM AUTHOR]

Trigonelline (TG; N-methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium Acinetobacter baylyi ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields (Z)-2-((N-methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into (E)-2-((N-methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available. [ABSTRACT FROM AUTHOR]

Abstract: Trans‐4‐Hydroxy‐ l‐proline (trans‐Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Bioconversion of l‐proline using recombinant strain with proline‐4‐hydroxylase (P4H) is a preferred biocatalytic process in the economical production of trans‐Hyp. In this study, a recombinant E. coli overexpressing hydroxylase (P4H), γ‐glutamyl kinase and glutamate‐semialdehyde dehydrogenase (ProBA) genes were constructed by knocking out the key genes in the metabolism. These key genes contained putA encoding proline dehydrogenase (PutA) in the l‐proline metabolism and other catalytic enzyme genes, sucAB encoding α‐ketoglutarate dehydrogenase (SucAB), aceAK encoding isocitratelyase (AceA) and isocitrate dehydrogenase kinase/phosphatase (AceK) in the TCA cycle. This recombinant strain coupled the synthetic pathway of trans‐Hyp with TCA cycle of the host strain. It inhibited the consumption of l‐proline completely and promoted the accumulation of 2‐oxoglutarate (2‐OG) as a co‐substrate, which realized the highest conversion of glucose to trans‐Hyp. A fed‐batch strategy was designed, capable of producing 31·0 g l−1trans‐Hyp from glucose. It provided a theoretical basis for commercial conversion of glucose to trans‐Hyp. Significance and Impact of the Study:Trans‐4‐Hydroxy‐ l‐proline (trans‐Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Unsatisfactory microbial bioconversion resulted in a low yield of trans‐Hyp. In this study, we blocked the unwanted blunting pathways of host strain and make the cell growth couple with the trans‐Hyp synthesis from glucose. Finally, a recombinant Escherichia coli with short‐cut and efficient trans‐Hyp biosynthetic pathway was obtained. It provided a theoretical basis for commercial production of trans‐Hyp. [ABSTRACT FROM AUTHOR]

It has been proposed that NADP+-specificity of isocitrate dehydrogenase (ICDH) evolved as an adaptation of microorganisms to grow on acetate as the sole source of carbon and energy. In Escherichia coli, changing the cofactor specificity of ICDH from NADP+ to NAD+ (cofactor swapping) decreases the growth rate on acetate. However, the metabolic basis of this phenotype has not been analyzed. In this work, we used constraint-based modeling to investigate the effect of the cofactor swapping of ICDH in terms of energy production, response of alternative sources of NADPH, and partitioning of fluxes between ICDH and isocitrate lyase (ICL) -a crucial bifurcation when the bacterium grows on acetate-. We generated E. coli strains expressing NAD+-specific ICDH instead of the native enzyme, and bearing the deletion of the NADPH-producing transhydrogenase PntAB. We measured their growth rate and acetate uptake rate, modeled the distribution of metabolic fluxes by Flux Balance Analysis (FBA), and quantified the specific activities of NADPH-producing dehydrogenases in central pathways. The cofactor swapping of ICDH led to one-third decrease in biomass yield, irrespective of the presence of PntAB. According to our simulations, the diminution in growth rate observed upon cofactor swapping could be explained by one-half decrease in the total production of NADPH and a lower availability of carbon for biosynthesis because of a change in the partition at the isocitrate bifurcation. Together with an increased total ATP production, this scenario resulted in a 10-fold increment in the flux of ATP not used for growing purposes. PntAB was identified as the primary NADPH balancing response, with the dehydrogenases of the oxidative branch of the Pentose Phosphate Pathway and the malic enzyme playing a role in its absence. We propose that in the context of E. coli growing on acetate, the NADP+-specificity of ICDH is a trait that impacts not only NADPH production, but also the efficient allocation of carbon and energy. [ABSTRACT FROM AUTHOR]

There is still no consensus on the true origin of fatal yellowing, one of the most important diseases affecting oil palm (Elaeis guineensis Jacq.) plantations. This study involved two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-UPLC-MSE) analyses to identify changes in protein profiles of oil palms affected by FY disease. Oil palm roots were sampled from two growing areas. Differential accumulation of proteins was assessed by comparing plants with and without symptoms and between plants at different stages of FY development. Most of the proteins identified with differential accumulation were those related to stress response and energy metabolism. The latter proteins include the enzymes alcohol dehydrogenase and aldehyde dehydrogenase, related to alcohol fermentation, which were identified in plants with and without symptoms. The presence of these enzymes suggests an anaerobic condition before or during FY. Transketolase, isoflavone reductase, cinnamyl alcohol dehydrogenase, caffeic acid 3-O-methyltransferase, S-adenosylmethionine synthase, aldehyde dehydrogenase and ferritin, among others, were identified as potential marker proteins and could be used to guide selection of FY-tolerant oil palm genotypes or to understand the source of this anomaly. When comparing different stages of FY, we observed high accumulation of alcohol dehydrogenase and other abiotic stress related-proteins at all disease stages. On the other hand, biological stress-related proteins were more accumulated at later stages of the disease. These results suggest that changes in abiotic factors can trigger FY development, creating conditions for the establishment of opportunistic pathogens. [ABSTRACT FROM AUTHOR]

Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the non-oxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production. [ABSTRACT FROM AUTHOR]

Glucose 6-phosphate dehydrogenase (G6PDH) is one of the most important dehydrogenases responsible for generating reduced NADPH for anabolism and is also the rate-limiting enzyme in the Entner-Doudoroff pathway. For in vitro biocatalysis, G6PDH must possess both high activity and good thermostability due to requirements of efficient use and low expense of biocatalyst. Here, we used directed evolution to improve thermostability of the highly active G6PDH from Zymomonas mobilis. Four generations of random mutagenesis and Petri-dish-based double-layer screening evolved the thermolabile wild-type enzyme to the thermostable mutant Mut 4-1, which showed a more than 124-fold increase in half-life time (t1/2) at 60 °C, a 3.4 °C increase in melting temperature (Tm), and a 5 °C increase in optimal temperature (Topt), without compromising the specific activity. In addition, the thermostable mutant was conducted to generate hydrogen from maltodextrin via in vitro synthetic biosystems (ivSB), gaining a more than 8-fold improvement of productivity rate with 76% of theoretical yield at 60 °C. Thus, the engineered G6PDH has been shown to effectively regenerate NADPH at high temperatures and will be applicable for NAD(P)H regeneration in numerous in vitro biocatalysis applications. [ABSTRACT FROM AUTHOR]

S-sulfhydration is a signalling pathway of hydrogen sulfide (H2S), which is suggested as an anti-atherogenic molecule that may protect against atherosclerosis. The identification of S-sulfhydrated proteins by proteomic approach could be a major step towards understanding the mechanisms of H2S in response to atherosclerosis. The present study studied targeted S-sulfhydrated proteins using the modified biotin switch method followed by matrix-assisted laser desorption/ionisation time of flight tandem mass spectrometry identification. The results showed that H2S can protect against atherosclerosis by reducing body weight gain and alleviating aortic plaque formation. In addition, H2S treatment can increase aortic protein S-sulfhydration. Seventy targeted S-sulfhydrated aortic proteins were identified, mainly involved in metabolism, stimulus response and biological regulation, as determined by gene ontology database analysis. H2S also induced S-sulfhydration of glutathione peroxidase 1 and further reduced lipid peroxidation and increased antioxidant defence in the aorta by prompting glutathione synthesis. Our data suggest that H2S is a cardiovascular-protective molecule that S-sulfhydrates a subset of proteins that are mainly responsible for lipid metabolism and exerts its cytoprotective effects to clear free radicals and inhibit oxidative stress through cysteine S-sulfhydration. [ABSTRACT FROM AUTHOR]

Reduced antimicrobial susceptibility due to resistance and tolerance has become a serious threat to human health. An approach to overcome this reduced susceptibility is the use of antibiotic adjuvants, also known as potentiators. These are compounds that have little or no antibacterial effect on their own but increase the susceptibility of bacterial cells towards antimicrobial agents. Baicalin hydrate, previously described as a quorum sensing inhibitor, is such a potentiator that increases the susceptibility of Burkholderia cenocepacia J2315 biofilms towards tobramycin. The goal of the present study is to elucidate the molecular mechanisms behind the potentiating activity of baicalin hydrate and related flavonoids. We first determined the effect of multiple flavonoids on susceptibility of B. cenocepacia J2315 towards tobramycin. Increased antibiotic susceptibility was most pronounced in combination with apigenin 7-O-glucoside and baicalin hydrate. For baicalin hydrate, also other B. cepacia complex strains and other antibiotics were tested. The potentiating effect was only observed for aminoglycosides and was both strain- and aminoglycoside-dependent. Subsequently, gene expression was compared between baicalin hydrate treated and untreated cells, in the presence and absence of tobramycin. This revealed that baicalin hydrate affected cellular respiration, resulting in increased reactive oxygen species production in the presence of tobramycin. We subsequently showed that baicalin hydrate has an impact on oxidative stress via several pathways including oxidative phosphorylation, glucarate metabolism and by modulating biosynthesis of putrescine. Furthermore, our data strongly suggest that the influence of baicalin hydrate on oxidative stress is unrelated to quorum sensing. Our data indicate that the potentiating effect of baicalin hydrate is due to modulating the oxidative stress response, which in turn leads to increased tobramycin-mediated killing. [ABSTRACT FROM AUTHOR]

The production of lignocellulosic-derived biofuels is a highly promising source of alternative energy, but it has been constrained by the lack of a microbial platform capable to efficiently degrade this recalcitrant material and cope with by-products that can be toxic to cells. Species that naturally grow in environments where carbon is mainly available as lignin are promising for finding new ways of removing the lignin that protects cellulose for improved conversion of lignin to fuel precursors. Enterobacter lignolyticus SCF1 is a facultative anaerobic Gammaproteobacteria isolated from tropical rain forest soil collected in El Yunque forest, Puerto Rico under anoxic growth conditions with lignin as sole carbon source. Whole transcriptome analysis of SCF1 during E.lignolyticus SCF1 lignin degradation was conducted on cells grown in the presence (0.1%, w/w) and the absence of lignin, where samples were taken at three different times during growth, beginning of exponential phase, mid-exponential phase and beginning of stationary phase. Lignin-amended cultures achieved twice the cell biomass as unamended cultures over three days, and in this time degraded 60% of lignin. Transcripts in early exponential phase reflected this accelerated growth. A complement of laccases, aryl-alcohol dehydrogenases, and peroxidases were most up-regulated in lignin amended conditions in mid-exponential and early stationary phases compared to unamended growth. The association of hydrogen production by way of the formate hydrogenlyase complex with lignin degradation suggests a possible value added to lignin degradation in the future. [ABSTRACT FROM AUTHOR]

Main conclusion : The physiological phenotype of potato tubers afflicted by zebra chip disease is characterized by increased oxidative stress metabolism and upregulation of systems for its mitigation. Starch catabolism and extensive buildup of reducing sugars render potatoes infected with zebra chip (ZC) pathogen ( Candidatus Liberibacter solanacearum) unsuitable for fresh market and processing into chips/fries. Here we show that the disease inflicts considerable oxidative stress, which likely constitutes a substantial sink for metabolic energy, resulting in increased respiration rate of afflicted tubers. In contrast to healthy tubers, tissue from diseased tubers had greater ability to reduce 2,3,5-triphenyl-tetrazolium chloride to formazan, indicating enhanced dehydrogenase activity, probable disease-induced changes in cellular redox potential, and increased respiratory activity. The respiration rate of diseased tubers (cv. Atlantic) was 2.4-fold higher than healthy tubers and this correlated with increased activities of glucose-6-phosphate and 6-phosphogluconate dehydrogenases, key enzymes responsible for synthesis of cytosolic reducing equivalents. Wound-induced NADPH oxidase activity was greater for ZC than healthy tubers, but the resulting superoxide was rapidly catabolized by higher superoxide dismutase activity in ZC tubers. Peroxidase, catalase, glutathione reductase and ascorbate free radical reductase activities were also higher in diseased tubers, as was malondialdehyde, a biomarker of peroxidative damage and oxidative stress. Upregulation of the glutathione-ascorbate pathway is a direct response to (and indicator of) oxidative stress, which consumes reducing equivalents (NADPH) to catabolize reactive oxygen species and maintain cellular redox homeostasis. ZC disease substantially altered the oxidative metabolism of tubers, resulting in a physiological phenotype defined by metabolic changes directed toward mitigating oxidative stress. Paradoxically, the increased respiration rate of ZC tubers, which fuels the metabolic pathways responsible for attenuating oxidative stress, likely also contributes to oxidative stress. [ABSTRACT FROM AUTHOR]

Cancer cells share several metabolic traits, including aerobic production of lactate from glucose (Warburg effect), extensive glutamine utilization and impaired mitochondrial electron flow. It is still unclear how these metabolic rearrangements, which may involve different molecular events in different cells, contribute to a selective advantage for cancer cell proliferation. To ascertain which metabolic pathways are used to convert glucose and glutamine to balanced energy and biomass production, we performed systematic constraint-based simulations of a model of human central metabolism. Sampling of the feasible flux space allowed us to obtain a large number of randomly mutated cells simulated at different glutamine and glucose uptake rates. We observed that, in the limited subset of proliferating cells, most displayed fermentation of glucose to lactate in the presence of oxygen. At high utilization rates of glutamine, oxidative utilization of glucose was decreased, while the production of lactate from glutamine was enhanced. This emergent phenotype was observed only when the available carbon exceeded the amount that could be fully oxidized by the available oxygen. Under the latter conditions, standard Flux Balance Analysis indicated that: this metabolic pattern is optimal to maximize biomass and ATP production; it requires the activity of a branched TCA cycle, in which glutamine-dependent reductive carboxylation cooperates to the production of lipids and proteins; it is sustained by a variety of redox-controlled metabolic reactions. In a K-ras transformed cell line we experimentally assessed glutamine-induced metabolic changes. We validated computational results through an extension of Flux Balance Analysis that allows prediction of metabolite variations. Taken together these findings offer new understanding of the logic of the metabolic reprogramming that underlies cancer cell growth. [ABSTRACT FROM AUTHOR]

A fluorene-degrading bacterium Paenibacillus sp. PRNK-6, capable of catabolizing 75% of fluorene (280 mg l −1 ) in 24 h, was isolated from PAHs contaminated soil. The metabolites formed were characterized by GC and GC–HRMS. The involvement of relevant enzymes, namely, fluorene mono-oxygenase, 9-hydroxyfluorene dehydrogenase, phthalate 4,5-dioxygenase, protocatechuate 3,4-dioxygenase, catechol 1,2-dioxygenase was demonstrated. Based on these results and metabolite-feeding experiments we propose a putative fluorene catabolic pathway in PRNK-6. The pathway for fluorene in strain PRNK-6 is: Fluorene → 9-fluorenol → 9-fluorenone → 1,2-dihydroxy-9-fluorenone → 2,3-dihydroxy indanone → 2-carboxybenzaldehyde → phthalate. Phthalate is catabolized through two pathways. One pathway involves the sequential conversion of phthalate to 4,5-dihydroxyphthalate and protocatechuate. In the second pathway phthalate is decarboxylated to benzoate, which is further converted to 4-hydroxybenzoate, protocatechuate and catechol sequentially. In either case, dead end products have not accumulated. [ABSTRACT FROM AUTHOR]

Release of arachidonic acid (AA) by cytoplasmic phospholipase A2 (cPLA2), followed by metabolism through cyclooxygenase-2 (COX-2) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH), results in the formation of the eicosanoids 11-oxo- and 15-oxo-eicosatetraenoic acid (oxo-ETE). Both 11-oxo- and 15-oxo-ETE have been identified in human biospecimens but their function and further metabolism is poorly described. The oxo-ETEs contain an α,β-unsaturated ketone and a free carboxyclic acid, and thus may form Michael adducts with a nucleophile or a thioester with the free thiol of Coenzyme A (CoA). To examine the potential for eicosanoid-CoA formation, which has not previously been a metabolic route examined for this class of lipids, we applied a semi-targeted neutral loss scanning approach following arachidonic acid treatment in cell culture and detected inducible long-chain acyl-CoAs including a predominant AA-CoA peak. Interestingly, a series of AA-inducible acyl-CoAs at lower abundance but higher mass, likely corresponding to eicosanoid metabolites, was detected. Using a targeted LC–MS/MS approach we detected the formation of CoA thioesters of both 11-oxo- and 15-oxo-ETE and monitored the kinetics of their formation. Subsequently, we demonstrated that these acyl-CoA species undergo up to four double bond reductions. We confirmed the generation of 15-oxo-ETE-CoA in human platelets via LC-high resolution MS. Acyl-CoA thioesters of eicosanoids may provide a route to generate reducing equivalents, substrates for fatty acid oxidation, and substrates for acyl-transferases through cPLA2-dependent eicosanoid metabolism outside of the signaling contexts traditionally ascribed to eicosanoid metabolites. [ABSTRACT FROM AUTHOR]

Peach leaf curl is a disease that affects the leaves of peach trees, and in severe cases all of the leaf can be similarly affected. This study investigated some effects of this disease on the metabolism of peach leaves in which all parts of the leaf were infected. These diseased leaves contained very little chlorophyll and performed little or no photosynthesis. Compared to uninfected leaves, diseased leaves possessed higher contents of fructose and especially glucose, but lowered contents of sucrose, sorbitol and especially starch. The activities of soluble acid invertase, neutral invertase, sorbitol dehydrogenase and sucrose synthase were all higher in diseased leaves, whereas, those of aldose-6-phosphate reductase and sucrose phosphate synthase were lower. The activities of hexokinase and fructokinase were little changed. In addition, immunblots showed that the contents of Rubisco and ADP-glucose phosphorylase were reduced in diseased leaves, whereas, the content of phosphoenolpyruvate carboxylase was increased. The results show that certain aspects of the metabolism of diseased leaves are similar to immature sink leaves. That is photosynthetic function is reduced, the leaf imports rather than exports sugars, and the contents of non-structural carbohydrates and enzymes involved in their metabolism are similar to sink leaves. Further, the effects of peach leaf curl on the metabolism of peach leaves are comparable to the effects of some other diseases on the metabolism of photosynthetic organs of other plant species. [ABSTRACT FROM AUTHOR]

Halophilic bacteria use a variety of osmoregulatory methods, such as the accumulation of one or more compatible solutes. The wide diversity of compounds that can act as compatible solute complicates the task of understanding the different strategies that halophilic bacteria use to cope with salt. This is specially challenging when attempting to go beyond the pathway that produces a certain compatible solute towards an understanding of how the metabolic network as a whole addresses the problem. Metabolic reconstruction based on genomic data together with Flux Balance Analysis (FBA) is a promising tool to gain insight into this problem. However, as more of these reconstructions become available, it becomes clear that processes predicted by genome annotation may not reflect the processes that are active in vivo. As a case in point, E. coli is unable to grow aerobically on citrate in spite of having all the necessary genes to do it. It has also been shown that the realization of this genetic potential into an actual capability to metabolize citrate is an extremely unlikely event under normal evolutionary conditions. Moreover, many marine bacteria seem to have the same pathways to metabolize glucose but each species uses a different one. In this work, a metabolic network inferred from genomic annotation of the halophilic bacterium Halomonas elongata and proteomic profiling experiments are used as a starting point to motivate targeted experiments in order to find out some of the defining features of the osmoregulatory strategies of this bacterium. This new information is then used to refine the network in order to describe the actual capabilities of H. elongata, rather than its genetic potential. [ABSTRACT FROM AUTHOR]

Lactobacillus reuteri, a heterofermentative bacterium, metabolizes glycerol via a Pdu (propanediol-utilization) pathway involving dehydration to 3-hydroxypropionaldehyde (3-HPA) followed by reduction to 1,3-propandiol (1,3-PDO) with concomitant generation of an oxidized cofactor, NAD+ that is utilized to maintain cofactor balance required for glucose metabolism and even for oxidation of 3-HPA by a Pdu oxidative branch to 3-hydroxypropionic acid (3-HP). The Pdu pathway is operative inside Pdu microcompartment that encapsulates different enzymes and cofactors involved in metabolizing glycerol or 1,2-propanediol, and protects the cells from the toxic effect of the aldehyde intermediate. Since L. reuteri excretes high amounts of 3-HPA outside the microcompartment, the organism is likely to have alternative alcohol dehydrogenase(s) in the cytoplasm for transformation of the aldehyde. In this study, diversity of alcohol dehydrogenases in Lactobacillus species was investigated with a focus on L. reuteri. Nine ADH enzymes were found in L. reuteri DSM20016, out of which 3 (PduQ, ADH6 and ADH7) belong to the group of iron-dependent enzymes that are known to transform aldehydes/ketones to alcohols. L. reuteri mutants were generated in which the three ADHs were deleted individually. The lagging growth phenotype of these deletion mutants revealed that limited NAD+/NADH recycling could be restricting their growth in the absence of ADHs. Notably, it was demonstrated that PduQ is more active in generating NAD+ during glycerol metabolism within the microcompartment by resting cells, while ADH7 functions to balance NAD+/NADH by converting 3-HPA to 1,3-PDO outside the microcompartment in the growing cells. Moreover, evaluation of ADH6 deletion mutant showed strong decrease in ethanol level, supporting the role of this bifuctional alcohol/aldehyde dehydrogenase in ethanol production. To the best of our knowledge, this is the first report revealing both internal and external recycling for cofactor homeostasis during 3-HPA conversion in L. reuteri. [ABSTRACT FROM AUTHOR]

Low temperature (LT) is one of the most important abiotic stresses that can significantly reduce crop yield. To gain insight into how Spirulina responds to LT stress, comprehensive physiological and proteomic analyses were conducted in this study. Significant decreases in growth and pigment levels as well as excessive accumulation of compatible osmolytes were observed in response to LT stress. An isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics approach was used to identify changes in protein abundance in Spirulina under LT. A total of 3,782 proteins were identified, of which 1,062 showed differential expression. Bioinformatics analysis indicated that differentially expressed proteins that were enriched in photosynthesis, carbohydrate metabolism, amino acid biosynthesis, and translation are important for the maintenance of cellular homeostasis and metabolic balance in Spirulina when subjected to LT stress. The up-regulation of proteins involved in gluconeogenesis, starch and sucrose metabolism, and amino acid biosynthesis served as coping mechanisms of Spirulina in response to LT stress. Moreover, the down-regulated expression of proteins involved in glycolysis, TCA cycle, pentose phosphate pathway, photosynthesis, and translation were associated with reduced energy consumption. The findings of the present study allow a better understanding of the response of Spirulina to LT stress and may facilitate in the elucidation of mechanisms underlying LT tolerance. [ABSTRACT FROM AUTHOR]

The whole-genome sequence of Bombella intestini LMG 28161T, an endosymbiotic acetic acid bacterium (AAB) occurring in bumble bees, was determined to investigate the molecular mechanisms underlying its metabolic capabilities. The draft genome sequence of B. intestini LMG 28161T was 2.02 Mb. Metabolic carbohydrate pathways were in agreement with the metabolite analyses of fermentation experiments and revealed its oxidative capacity towards sucrose, D-glucose, D-fructose and D-mannitol, but not ethanol and glycerol. The results of the fermentation experiments also demonstrated that the lack of effective aeration in small-scale carbohydrate consumption experiments may be responsible for the lack of reproducibility of such results in taxonomic studies of AAB. Finally, compared to the genome sequences of its nearest phylogenetic neighbor and of three other insect associated AAB strains, the B. intestini LMG 28161T genome lost 69 orthologs and included 89 unique genes. Although many of the latter were hypothetical they also included several type IV secretion system proteins, amino acid transporter/permeases and membrane proteins which might play a role in the interaction with the bumble bee host. [ABSTRACT FROM AUTHOR]

Yarrowia lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content. Here, we engineered Y. lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80 g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials. [ABSTRACT FROM AUTHOR]

Metabolic dysfunction is well-documented in Huntington’s disease (HD). However, the link between the mutant huntingtin (mHTT) gene and the pathology is unknown. The tricarboxylic acid (TCA) cycle is the main metabolic pathway for the production of NADH for conversion to ATP via the electron transport chain (ETC). The objective of this study was to test for differences in enzyme activities, mRNAs and protein levels related to the TCA cycle between lymphoblasts from healthy subjects and from patients with HD. The experiments utilize the advantages of lymphoblasts to reveal new insights about HD. The large quantity of homogeneous cell populations permits multiple dynamic measures to be made on exactly comparable tissues. The activities of nine enzymes related to the TCA cycle and the expression of twenty-nine mRNAs encoding for these enzymes and enzyme complexes were measured. Cells were studied under baseline conditions and during metabolic stress. The results support our recent findings that the activities of the pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase (SDH) are elevated in HD. The data also show a large unexpected depression in MDH activities. Furthermore, message levels for isocitrate dehydrogenase 1 (IDH1) were markedly increased in in HD lymphoblasts and were responsive to treatments. The use of lymphoblasts allowed us to clarify that the reported decrease in aconitase activity in HD autopsy brains is likely due to secondary hypoxic effects. These results demonstrate the mRNA and enzymes of the TCA cycle are critical therapeutic targets that have been understudied in HD. [ABSTRACT FROM AUTHOR]

Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70–4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes. [ABSTRACT FROM AUTHOR]