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The opportunistic human pathogen Acinetobacter baumannii can grow with carnitine but its metabolism, regulation and role in virulence remained elusive. Recently, we identified a carnitine transporter encoded by a gene closely associated with potential carnitine degradation genes. Among those is a gene coding for a putative d-malate dehydrogenase (Mdh). Deletion of the mdh gene led to a loss of growth with carnitine but not l-malate; growth with d-malate was strongly reduced. Therefore, it is hypothesized that d-malate is formed during carnitine oxidation and further oxidized to CO

Euglena gracilis produces ATP in the anaerobic mitochondria with concomitant wax ester formation, and NADH is essential for ATP formation and fatty acid synthesis in the mitochondria. This study demonstrated that mitochondrial cofactor conversion by nicotinamide nucleotide transhydrogenase (NNT), converting NADPH/NAD+ to NADP+ /NADH, is indispensable for sustaining anaerobic metabolism. Silencing of NNT genes significantly decreased wax ester production and cellular viability during anaerobiosis but had no such marked effects under aerobic conditions. An analogous phenotype was observed in the silencing of the gene encoding a mitochondrial NADP+ -dependent malic enzyme. These results suggest that the reducing equivalents produced in glycolysis are shuttled to the mitochondria as malate, where cytosolic NAD+ regeneration is coupled with mitochondrial NADPH generation.

Long non–coding RNA AC020978 (lncRNA AC020978) is an oncogenic regulator of non–small cell lung cancer (NSCLC). However, the function of AC020978 in regulating NSCLC metastasis and the potential molecular mechanism remains largely unknown. In this study, we evaluated the expression levels of AC020978 in a series of NSCLC tissues using FISH assays and found that higher AC020978 expression levels were closely associated with metastasis and unfavorable prognosis. Functional studies showed that AC020978 promoted NSCLC migration and invasion both in vitro and in vivo. Further investigation demonstrated that AC020978 interacted with malate dehydrogenase 2 (MDH2) and maintained MDH2 stability. Knockdown of MDH2 weakened the facilitating effect on cell metastasis and 2‐hydroxyglutarate (2‐HG) metabolism in AC020978‐overexpressed NSCLC cells. RNA sequencing, bioinformatic analysis, and western blotting revealed that AC020978 was associated with the AKT signaling pathway. Taken together, our findings revealed that AC020978 might serve as a prognostic biomarker and activate the AKT pathway by stabilizing MDH2, leading to metastasis and progression of NSCLC., We identified the metastasis‐related function of lncRNA AC020978 in NSCLC, which directly interacts with MDH2 and promotes migration and invasion of lung cancer through the AC020978/MDH2/AKT axis.

The use of nicotinamide cytosine dinucleotide (NCD), a biocompatible nicotinamide adenosine dinucleotide (NAD) analogue, is of great scientific and biotechnological interest. Several redox enzymes have been devised to favor NCD, and have been successfully applied in creating NCD-dependent redox systems. However, molecular interactions between cofactor and protein have still to be disclosed in order to guide further engineering efforts. Here we report the structural analysis of an NCD-favoring malic enzyme (ME) variant derived from Escherichia coli. The X-ray crystal structure data revealed that the residues located at position 346 and 401 in ME acted as the "gatekeepers" of the adenine moiety binding cavity. When Arg346 was substituted with either acidic or aromatic residues, the corresponding mutants showed substantially reduced NCD preference. Inspired by these observations, we generated Lactobacillus helveticus derived d-lactate dehydrogenase variants at Ile177, the counterpart to Arg346 in ME, and found a similar trend in terms of cofactor preference changes. As many NAD-dependent oxidoreductases share key structural features, our results provide guidance for protein engineering to obtain more NCD-favoring variants.

Background Ferulic acid (FA) is a common polyphenolic compound. The purpose of this study was to explore the effect of dietary FA supplementation on growth performance and muscle fiber type conversion in weaned piglets. In this study, eighteen 21-day-old DLY (Duroc × Landrace × Yorkshire) weaned piglets were randomly divided into control, 0.05% FA, and 0.45% FA groups. Results Our study showed that dietary FA supplementation had no effect on growth performance, but it could upregulate the expression of slow myosin heavy chain (MyHC) protein, increase the activities of succinic dehydrogenase and malate dehydrogenase, and downregulate the expression of fast MyHC protein. Dietary FA supplementation also increased the expression levels of phosphorylated AMP-activated protein kinase, sirtuin 1 (Sirt1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), myocyte enhancer factor 2C, and troponin I-SS, increased the proportion of slow-twitch fiber, and decreased the proportion of fast-twitch fiber. In addition, our results showed that dietary FA supplementation increased the messenger RNA abundance of mitochondrial nuclear transcription genes, including ATP synthase membrane subunit c locus 1, cytochrome oxidase subunit 1, nuclear respiratory factor 1, mitochondrial transcription factor A, mitochondrial transcription factor B1, and cytochrome c. Conclusion We provided the first evidence that FA could promote muscle fiber type conversion from fast-twitch to slow-twitch via the Sirt1/AMP-activated protein kinase/PGC-1α signaling pathway and could improve the mitochondrial function in weaned piglets. This means that FA can be used as a dietary supplement to improve the quality of pork. © 2021 Society of Chemical Industry.

Leishmania has a remarkable ability to proliferate under widely fluctuating levels of essential nutrients, such as glucose. For this, the parasite is heavily dependent on its gluconeogenic machinery. One perplexing aspect of gluconeogenesis in Leishmania is the lack of the crucial gene for pyruvate carboxylase (PC). PC-catalyzed conversion of pyruvate to oxaloacetate is a key entry point through which gluconeogenic amino acids are funneled into this pathway. The absence of PC in Leishmania thus raises question about the mechanism of pyruvate entry into the gluconeogenic route. In the present study, we report that this task is accomplished in Leishmania major through a novel functional partnership between its mitochondrial malic enzyme (LmME) and carbonic anhydrase 1 (LmCA1). Using a combination of pharmacological inhibition studies with genetic manipulation, we show that both of these enzymes are necessary for promoting gluconeogenesis and supporting parasite growth under glucose-limiting conditions. Functional cross-talk between LmME and LmCA1 was evident when it was observed that the growth retardation caused by inhibition of any one of these enzymes could be protected to a significant extent by overexpressing the other enzyme. We also found that, although LmCA1 exhibited constitutive expression, the LmME protein level was strongly upregulated under low glucose conditions. Notably, both LmME and LmCA1 were found to be important for survival of Leishmania amastigotes within host macrophages. Taken together, our results indicate that LmCA1 by virtue of its CO2 concentrating ability stimulates LmME-catalyzed pyruvate carboxylation, thereby driving gluconeogenesis through the pyruvate-malate-oxaloacetate bypass pathway. Additionally, our study establishes LmCA1 and LmME as promising therapeutic targets.

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways in nature. Oxygenic photoautotrophic bacteria, cyanobacteria, have an unusual TCA cycle. The TCA cycle in cyanobacteria contains two unique enzymes that are not part of the TCA cycle in other organisms. In recent years, sustainable metabolite production from carbon dioxide using cyanobacteria has been looked at as a means to reduce the environmental burden of this gas. Among cyanobacteria, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) is an optimal host for sustainable metabolite production. Recently, metabolite production using the TCA cycle in Synechocystis 6803 has been carried out. Previous studies revealed that the branch point of the oxidative and reductive TCA cycles, oxaloacetate metabolism, plays a key role in metabolite production. However, the biochemical mechanisms regulating oxaloacetate metabolism in Synechocystis 6803 are poorly understood. Concentrations of oxaloacetate in Synechocystis 6803 are extremely low, such that in vivo analysis of oxaloacetate metabolism does not seem realistic. Therefore, using purified enzymes, we reconstituted oxaloacetate metabolism in Synechocystis 6803 in vitro to reveal the regulatory mechanisms involved. Reconstitution of oxaloacetate metabolism revealed that pH, Mg2+ and phosphoenolpyruvate are important factors affecting the conversion of oxaloacetate in the TCA cycle. Biochemical analyses of the enzymes involved in oxaloacetate metabolism in this and previous studies revealed the biochemical mechanisms underlying the effects of these factors on oxaloacetate conversion. In addition, we clarified the function of two l-malate dehydrogenase isozymes in oxaloacetate metabolism. These findings serve as a basis for various applications of the cyanobacterial TCA cycle.

This article presents a personal and critical review of the history of the malate–aspartate shuttle (MAS), starting in 1962 and ending in 2020. The MAS was initially proposed as a route for the oxidation of cytosolic NADH by the mitochondria in Ehrlich ascites cell tumor lacking other routes, and to explain the need for a mitochondrial aspartate aminotransferase (glutamate oxaloacetate transaminase 2 [GOT2]). The MAS was soon adopted in the field as a major pathway for NADH oxidation in mammalian tissues, such as liver and heart, even though the energetics of the MAS remained a mystery. Only in the 1970s, LaNoue and coworkers discovered that the efflux of aspartate from mitochondria, an essential step in the MAS, is dependent on the proton‐motive force generated by the respiratory chain: for every aspartate effluxed, mitochondria take up one glutamate and one proton. This makes the MAS in practice uni‐directional toward oxidation of cytosolic NADH, and explains why the free NADH/NAD ratio is much higher in the mitochondria than in the cytosol. The MAS is still a very active field of research. Most recently, the focus has been on the role of the MAS in tumors, on cells with defects in mitochondria and on inborn errors in the MAS. The year 2019 saw the discovery of two new inborn errors in the MAS, deficiencies in malate dehydrogenase 1 and in aspartate transaminase 2 (GOT2). This illustrates the vitality of ongoing MAS research.

Summary Changes in carbohydrates and organic acids largely determine the palatability of edible tissues of horticulture crops. Elucidating the potential molecular mechanisms involved in the change in carbohydrates and organic acids, and their temporal and spatial crosstalk are key steps in understanding fruit developmental processes. Here, we used apple (Malus domestica Borkh.) as research materials and found that MdbHLH3, a basic helix–loop–helix transcription factor (bHLH TF), modulates the accumulation of malate and carbohydrates. Biochemical analyses demonstrated that MdbHLH3 directly binds to the promoter of MdcyMDH that encodes an apple cytosolic NAD‐dependent malate dehydrogenase, activating its transcriptional expression, thereby promoting malate accumulation in apple fruits. Additionally, MdbHLH3 overexpression increased the photosynthetic capacity and carbohydrate levels in apple leaves and also enhanced the carbohydrate accumulation in fruits by adjusting carbohydrate allocation from sources to sinks. Overall, our findings provide new insights into the mechanism of how the bHLH TF MdbHLH3 modulates the fruit quality. It directly regulates the expression of cytosolic malate dehydrogenase MdcyMDH to coordinate carbohydrate allocation and malate accumulation in apple.

The citric acid cycle, also known as the Krebs cycle, plays an integral role in cellular metabolism and aerobic respiration. Mutations in genes encoding the citric acid cycle enzymes succinate dehydrogenase, fumarate hydratase and malate dehydrogenase all predispose to hereditary tumour syndromes. The succinate dehydrogenase enzyme complex (SDH) couples the oxidation of succinate to fumarate in the citric acid cycle and the reduction of ubiquinone to ubiquinol in the electron transport chain. A loss of function in the succinate dehydrogenase (SDH) enzyme complex is most commonly caused by an inherited mutation in one of the four SDHx genes (SDHA, SDHB, SDHC and SDHD). This mechanism was first implicated in familial phaeochromocytoma and paraganglioma. However, over the past two decades the spectrum of tumours associated with SDH deficiency has been extended to include gastrointestinal stromal tumours (GIST), renal cell carcinoma (RCC) and pituitary adenomas. The aim of this review is to describe the extended tumour spectrum associated with SDHx gene mutations and to consider how functional tests may help to establish the role of SDHx mutations in new or unexpected tumour phenotypes.

Background Camptothecin (CPT) and matrine (MAT) have potential as botanical pesticides against several pest species. However, the mechanisms of metabolic and physiological changes in pests induced by CPT and MAT are unknown. In this study, a toxicological test, an NMR-based metabolomic study, an enzymatic test, and an RT quantitative PCR (RT-qPCR) experiment were all conducted to examine the effect of CPT and MAT on Spodoptera litura. Results CPT (0.5-1%) exerted high toxicity against larvae of S. litura and caused growth stagnation and high mortality of larvae. A variety of metabolites were significantly influenced by 0.5% CPT, including several energy-related metabolites such as trehalose, lactate, succinate, citrate, malate, and fumarate. In contrast, MAT showed low toxicity against larvae and induced almost no changes in hemolymph metabolites of S. litura. Enzymatic tests showed that trehalase activity was significantly decreased in larvae after feeding with 0.5% CPT. RT-qPCR showed that the transcription levels of alanine aminotransferase, malate dehydrogenase, and isocitrate dehydrogenase were decreased while lactate dehydrogenase was increased in the 0.5% CPT-treated group. Conclusions These data indicate that one of the important mechanisms of CPT against S. litura larvae is via the inhibition of trehalose hydrolysis and glycolysis. Our findings also suggest that CPT exhibits a stronger toxicological effect than MAT against S. litura, which provides basic information for the application of CPT in the control of S. litura or other lepidoptera pests.

Background Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum threatens oilseed rape cultivation, and the emergence of fungicide-resistant strains has led to control failures worldwide. Identifying novel chemical alternatives with different modes of action and high antifungal activities is thus crucial. Herein we evaluated the antifungal effects of 3-(2-pyridyl)methyl-2-(4-chlorphenyl)imino- thiazolidine (PMAS) on S. sclerotiorum to determine its efficacy for SSR management. Results PMAS had an inhibitory effect on mycelial growth; the EC50 values were 17.83 and 21.15 μg mL-1 for the carbendazim-susceptible strain Ss01 and carbendazim-resistant strain Hm25, respectively. PMAS treatment changed the color of inhibited mycelia to green, and the hyphae were sustained in the undifferentiated stage. Cysteine supplementation made this green color disappear, whereas methionine enhanced the color. Moreover, PMAS treatment markedly inhibited oxalic acid biogenesis, increased free thiol content in mycelia, and weakened the activities of oxaloacetase and malate dehydrogenase, but had little effect on the activity of glyoxylate dehydrogenase. Cysteine could reverse the inhibitory effects of PMAS on mycelial morphogenesis and biochemical constituents, except thiol production. In the pot-culture experiment, PMAS showed a good protective effect, with the control efficacy being >91% on SSR. Conclusion PMAS appears to be an effective fungicide for SSR management. © 2020 Society of Chemical Industry.

Phosphoenolpyruvate carboxylase (PEPC) is the second major carbon-fixing enzyme in photoautotrophic organisms. PEPC is required for the synthesis of amino acids of the glutamate and aspartate family by replenishing the TCA cycle. Furthermore, in cyanobacteria, PEPC, together with malate dehydrogenase and malic enzyme, forms a metabolic shunt for the synthesis of pyruvate from PEP. During this process, CO2 is first fixed and later released again. Due to its central metabolic position, it is crucial to fully understand the regulation of PEPC. Here, we identify PEPC from the cyanobacterium Synechocystis sp. PCC 6803 (PEPC) as a novel interaction partner for the global signal transduction protein PII . In addition to an extensive characterization of PEPC, we demonstrate specific PII -PEPC complex formation and its enzymatic consequences. PEPC activity is tuned by the metabolite-sensing properties of PII : Whereas in the absence of PII, PEPC is subjected to ATP inhibition, it is activated beyond its basal activity in the presence of PII . Furthermore, PII -PEPC complex formation is inhibited by ADP and PEPC activation by PII -ATP is mitigated in the presence of 2-OG, linking PEPC regulation to the cell's global carbon/nitrogen status. Finally, physiological relevance of the in vitro measurements was proven by metabolomic analyses of Synechocystis wild-type and PII -deficient cells.

d -Phenyllactate (PLA) is a component of the selective Gq protein inhibitor and nonribosomal cyclic depsipeptide FR900359 (FR). Here we perform a detailed biochemical investigation of PLA biosynthesis and its incorporation into the natural product FR. The enzyme FrsC, member of the lactate/malate dehydrogenase superfamily, was shown to catalyze the formation of l -PLA from phenylpyruvate. FrsC was kinetically characterized and its substrate specificity determined. Incorporation of l -PLA was probed by assaying the adenylation domain FrsE-A3 and feeding studies to a Chromobacterium vaccinii ΔfrsC mutant, confirming preferred activation of l -PLA followed by on-line epimerisation to d -PLA. Finally, detailed bioinformatic analyses of FrsC revealed its close relation to malate dehydrogenases from primary metabolism and suggest extensions in the substrate binding loop to be responsible for its adaptation to accept larger aromatic substrates with high specificity.

A diet based on sunflower cake for lambs was assayed in order to reuse biodiesel industrial by‐products with the aim of reducing livestock costs and evaluating their influence on meat quality. To achieve these goals, sixteen male lambs were fed diets containing different levels of sunflower cake (control, 5%, 10% and 15%). Afterwards, their semimembranosus muscles were analysed by two‐dimensional electrophoresis coupled to mass spectrometry and their functional protein association was examined using STRING. Structural and metabolic proteins in the lambs’ proteomes changed significantly according to their diet. Fifteen proteins showed significant changes caused by the inclusion of sunflower cake, and the most differentially abundant structural proteins were detected in 2‐DE gels from the lambs. Differentially abundant metabolic proteins such as ENO3 (enolase 3), MDH1 (malate dehydrogenase) and ALDH1A1 (retinal dehydrogenase) have been proposed as biomarkers of quality parameters in other species.

The C12 specific oxidation of hydroxysteroids is an essential reaction required for the preparation of pharmaceutical ingredients like ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA), which can be synthesized by Wolff-Kishner reduction of the obtained 12-oxo-hydroxysteroids. 12α-hydroxysteroid dehydrogenases (12α-HSDHs) have been shown to perform this reaction with high yields, under mild conditions and without the need of protection and deprotection steps, required in chemical synthesis. Here, the recombinant expression and biochemical characterization of the nicotinamide adenine dinucleotide (NAD + )-dependent HSDH from Eggerthella lenta (El12α-HSDH) are reported. This enzyme shows comparable properties with the well-known nicotinamide adenine dinucleotide phosphate (NADP + )-dependent enzyme from Clostridium sp. 48–50. In order to perform a viable and atom efficient enzymatic hydroxysteroid oxidation, NAD(P)H oxidase (NOX) was employed as cofactor regeneration system: NOX uses oxygen (O 2 ) as sacrificial substrate and produces only water as side product. 10 mM of cholic acid was fully and selectively converted to 12-oxo-CDCA in 24 h. The possibility to employ this system on UCA and 7-oxo-deoxycholic acid (7-oxo-DCA) as substrates was additionally investigated. The performance of the El12α-HSDH was evaluated also in combination with a “classical” regeneration system (oxaloacetate/malate dehydrogenase) showing full conversion in 4 h. Finally, the feasibility of a catalytic aerobic-NAD + -dependent enzymatic oxidation was shown on a preparative scale (oxidation of CA to 12-oxo-CDCA) employing the El12α-HSDH-NOX system in a segmented-flow-reactor. (Figure presented.).

Summary Starch is the most important form of energy storage in cereal crops. Many key enzymes involved in starch biosynthesis have been identified. However, the molecular mechanisms underlying the regulation of starch biosynthesis are largely unknown. In this study, we isolated a novel floury endosperm rice (Oryza sativa) mutant flo16 with defective starch grain (SG) formation. The amylose content and amylopectin structure were both altered in the flo16 mutant. Map‐based cloning and complementation tests demonstrated that FLO16 encodes a NAD‐dependent cytosolic malate dehydrogenase (CMDH). The ATP contents were decreased in the mutant, resulting in significant reductions in the activity of starch synthesis‐related enzymes. Our results indicated that FLO16 plays a critical role in redox homeostasis that is important for compound SG formation and subsequent starch biosynthesis in rice endosperm. Overexpression of FLO16 significantly improved grain weight, suggesting a possible application of FLO16 in rice breeding. These findings provide a novel insight into the regulation of starch synthesis and seed development in rice.

A previous study demonstrated that leucine upregulates the slow myosin heavy chain mRNA expression in C2C12 cells. However, the role of leucine in slow-twitch muscle fibers expression and mitochondrial function of porcine skeletal muscle satellite cells as well as its mechanism remain unclear. In this study, porcine skeletal muscle satellite cells cultured in differentiation medium were treated with 2 mM leucine for 3 days. Sirt1 inhibitor EX527, AMPK inhibitor compound C, and AMPKα1 siRNA were used to examine its underlying mechanism. Here we showed that leucine increased slow-twitch muscle fibers and mitochondrial function-related gene expression, as well as increased succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. Moreover, leucine increased the protein levels of Sirt1 and phospho-AMPK. We also found that AMPKα1 siRNA, AMPK inhibitor compound C, or Sirt1 inhibitor EX527 attenuated the positive effect of leucine on slow-twitch muscle fibers and mitochondrial function-related gene expression. Finally, we showed that Sirt1 was required for leucine-induced AMPK activation. Our results provide, for the first time, evidence that leucine induces slow-twitch muscle fibers expression and improves mitochondrial function through Sirt1/AMPK signaling pathway in porcine skeletal muscle satellite cells.

Malate valves act as powerful systems for balancing the ATP/NAD(P)H ratio required in various subcellular compartments in plant cells. As components of malate valves, isoforms of malate dehydrogenases (MDHs) and dicarboxylate translocators catalyse the reversible interconversion of malate and oxaloacetate and their transport. Depending on the co‐enzyme specificity of the MDH isoforms, either NADH or NADPH can be transported indirectly. Arabidopsis thaliana possesses nine genes encoding MDH isoenzymes. Activities of NAD‐dependent MDHs have been detected in mitochondria, peroxisomes, cytosol and plastids. In addition, chloroplasts possess a NADP‐dependent MDH isoform. The NADP‐MDH as part of the ‘light malate valve’ plays an important role as a poising mechanism to adjust the ATP/NADPH ratio in the stroma. Its activity is strictly regulated by post‐translational redox‐modification mediated via the ferredoxin‐thioredoxin system and fine control via the NADP +/NADP(H) ratio, thereby maintaining redox homeostasis under changing conditions. In contrast, the plastid NAD‐MDH (‘dark malate valve’) is constitutively active and its lack leads to failure in early embryo development. While redox regulation of the main cytosolic MDH isoform has been shown, knowledge about regulation of the other two cytosolic MDHs as well as NAD‐MDH isoforms from peroxisomes and mitochondria is still lacking. Knockout mutants lacking the isoforms from chloroplasts, mitochondria and peroxisomes have been characterised, but not much is known about cytosolic NAD‐MDH isoforms and their role in planta. This review updates the current knowledge on MDH isoforms and the shuttle systems for intercompartmental dicarboxylate exchange, focusing on the various metabolic functions of these valves.

We previously engineered Escherichia coli to overproduce medium- to long-chain saturated and monounsaturated methyl ketones, which could potentially be applied as diesel fuel blending agents or in the flavor and fragrance industry. Recent efforts at strain optimization have focused on cofactor balance, as fatty acid-derived pathways face the systematic metabolic challenge of net NADPH consumption (in large part, resulting from the key fatty acid biosynthetic enzyme FabG [β-ketoacyl-ACP reductase]) and net NADH production. In this study, we attempted to mitigate cofactor imbalance by heterologously expressing NADH-dependent, rather than NADPH-dependent, versions of FabG identified in previous studies. Of the four NADH-dependent versions of FabG tested in our previously best-reported methyl ketone-producing strain (EGS1895), the version from Acholeplasma laidlawii (Al_FabG) showed the greatest increase in methyl ketone yield in shake flasks (35-75% higher than for an RFP negative-control strain, depending on sugar loading). An improved strain (EGS2920) attained methyl ketone titers during fed-batch fermentation of 5.4 ± 0.5 g/L, which were, on average, ca. 40% greater than those for the base strain (EGS1895) under fermentation conditions optimized in this study. Shotgun proteomic data for strains EGS2920 and EGS1895 during fed-batch fermentation were consistent with the goal of alleviating NADPH limitation through expression of Al_FabG. For example, relative to strain EGS1895, strain EGS2920 significantly upregulated glucose-6-phosphate isomerase (directing flux into glycolysis rather than the NADPH-producing pentose phosphate pathway) and downregulated MaeB (a NADP+ -dependent malate dehydrogenase). Overall, the results suggest that heterologous expression of NADH-dependent FabG in E. coli may improve sustained production of fatty acid-derived renewable fuels and chemicals.

This experiment was conducted to clarify the nutritional functions of rice and phytase addition for broiler chicks. Thirty-six 7-day-old male chicks (ROSS 308 strain) were assigned to one of the four treatment groups: corn- or rice-based diet groups and each diet with added phytase (2000 phytase units/kg diet) groups (corn + P or rice + P groups). The non-phytate phosphorus (npP) content in the diets with added phytase was approximately half of the requirement. Body weight gain and feed intake in the rice group was significantly higher than those in the corn group. Breast and thigh muscle weights and nitrogen retention in the rice group were significantly higher than that in the corn group. Although the efficiency of phosphorus retention (%) in the corn + P group was significantly higher than that in the corn group, no significant difference was observed between the rice and rice + P groups. Liver threonine dehydrogenase activity in the corn group was significantly higher than in the other three groups. These results indicate that rice is superior to corn as a starter diet in broiler chicks, and that phytase action in the rice-based diet was less than that in the corn-based diet.

Malate dehydrogenase (MDH) and citrate synthase (CS) are two pacemaking enzymes involved in the tricarboxylic acid (TCA) cycle. Oxaloacetate (OAA) molecules are the intermediate substrates that are transferred from the MDH to CS to carry out sequential catalysis. It is known that, to achieve a high flux of intermediate transport and reduce the probability of substrate leaking, a MDH-CS metabolon forms to enhance the OAA substrate channeling. In this study, we aim to understand the OAA channeling within possible MDH-CS metabolons that have different structural orientations in their complexes. Three MDH-CS metabolons from native bovine, wild-type porcine, and recombinant sources, published in recent work, were selected to calculate OAA transfer efficiency by Brownian dynamics (BD) simulations and to study, through electrostatic potential calculations, a possible role of charges that drive the substrate channeling. Our results show that an electrostatic channel is formed in the metabolons of native bovine and recombinant porcine enzymes, which guides the oppositely charged OAA molecules passing through the channel and enhances the transfer efficiency. However, the channeling probability in a suggested wild-type porcine metabolon conformation is reduced due to an extended diffusion length between the MDH and CS active sites, implying that the corresponding arrangements of MDH and CS result in the decrease of electrostatic steering between substrates and protein surface and then reduce the substrate transfer efficiency from one active site to another.

Reversible lysine acetylation is one of the most widely distributed post‐translational modifications; it is involved in a variety of biological processes and can be found in all three domains of life. Acetyltransferases and deacetylases work coordinately to control levels of protein acetylation. In this work, we applied the genetic code expansion strategy to site‐specifically incorporate N e‐thioacetyl‐l‐lysine (TAcK) as an analog of N e‐acetyl‐l‐lysine (AcK) into green fluorescent protein and malate dehydrogenase in Escherichia coli. We showed that TAcK could serve as an ideal functional mimic for AcK. It could also resist the bacterial sirtuin‐type deacetylase CobB. Thus, genetic incorporation of TAcK as a non‐deacetylatable analog of AcK into proteins will facilitate in vivo studies of protein acetylation.

High-throughput analysis of genomic and proteomic information lead to large numbers of target molecules. The bottlenecks are generally subsequent verification steps, which require development of efficient screening tools. Protein–protein and protein–ligand interactions constitute the basis of most biological processes in living organisms. In general, proteins are labeled with fluorescent dyes to enhance sensitivities and the fluorescence changes are widely utilized for determining protein–protein and protein–ligand bindings. This study is to demonstrate that the intrinsic fluorescence of proteins by tryptophan and tyrosine and its change accompanied by ligand binding is sufficiently sensitive for studying protein–ligand interactions. Malate dehydrogenase catalyzes inter-conversion of oxaloacetate and NADH to malate and NAD+, respectively. Malate did not change the fluorescence of malate dehydrogenase. But, the dissociation constants (KD) for NADH and NAD+ could be measured by fluorescence changes and they are comparable to KM determined by enzyme kinetics. The high KD (1.5 mM) for oxaloacetate agreed well with the ordered Bi Bi mechanism of malate dehydrogenase, in which NADH bound first and generated a binding site for oxaloacetate. The dissociation constant for cyclic di-GMP to malate dehydrogenase was also determined by fluorescence changes and it was comparable to that collected from computational molecular docking analysis. The KD for cyclic di-GMP to methionine aminopeptidase, glycerol dehydrogenase, and peptidase B could also be determined by intrinsic fluorescence changes as 58, 58, 52 μM, respectively. These results suggest that the intrinsic fluorescence of proteins by tryptophan and tyrosine is sensitive enough to determine protein–ligand interactions. It provides a label-free, quick-and-easy tool for screening the ligands to proteins.

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa. Fil: Breininger, Elizabeth. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina Fil: Dubois, D.. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina Fil: Pereyra, Vanina Eliana. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina Fil: Rodriguez, P. C.. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina Fil: Satorre, María Mercedes. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina Fil: Cetica, Pablo Daniel. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina

Glyoxalase II, the second of 2 enzymes in the glyoxalase system, is a hydroxyacylglutathione hydrolase that catalyses the hydrolysis of S-d-lactoylglutathione to form d-lactic acid and glutathione, which is released from the active site. The tripeptide glutathione is the major sulfhydryl antioxidant and has been shown to control several functions, including S-glutathionylation of proteins. S-Glutathionylation is a way for the cells to store reduced glutathione during oxidative stress, or to protect protein thiol groups from irreversible oxidation, and few enzymes involved in protein S-glutathionylation have been found to date. In this work, the enzyme glyoxalase II and its substrate S-d-lactoylglutathione were incubated with malate dehydrogenase or with actin, resulting in a glutathionylation reaction. Glyoxalase II was also submitted to docking studies. Computational data presented a high propensity of the enzyme to interact with malate dehydrogenase or actin through its catalytic site and further in silico investigation showed a high folding stability of glyoxalase II toward its own reaction product glutathione both protonated and unprotonated. This study suggests that glyoxalase II, through a specific interaction of its catalytic site with target proteins, could be able to perform a rapid and specific protein S-glutathionylation using its natural substrate S-d-lactoylglutathione. SIGNIFICANCE This article reports for the first time a possible additional role of Glo2 that, after interacting with a target protein, is able to promote S-glutathionylation using its natural substrate SLG, a glutathione derived compound. In this perspective, Glo2 can play a new important regulatory role inS-glutathionylation, acquiring further significance in cellular post-translational modifications of proteins.

The Malate Dehydrogenase CUREs Community (MCC) project, funded by a grant from the National Science Foundation, involves protein-centric, Course Based Undergraduate Research Experiences, CUREs, foc...

Sequential enzymes involved in a metabolic pathway are often found in specific and dynamic protein-protein interactions. The assembly of proteins into this metabolon allow the efficient transfer of substrate between adjacent proteins increasing the throughput and decreasing the potential for side metabolic reactions to occur between competing enzymes. One such pairing is the malate dehydrogenase (MDH) and citrate synthase (CS). In the forward direction of the tricarboxylic cycle, the oxidation of malate to OAA catalyzed by MDH is thermodynamically unfavorable. Coupling and potentially direct shuttling of product-substrate affords the cycle to continue. The interaction site between mitochondrial MDH and CS has been partially identified via computational docking and cross-linking studies. However, the domains and key residues of interaction and their impact on the actions and affinity of various isoforms of MDH and CS have not been identified. Using two biophysical techniques, protein thermal melts and surf...

Objective Genetic generalized epilepsy (GGE) consists of epileptic syndromes with overlapping symptoms and is considered to be largely genetic. Previous cosegregation and association studies have pointed to malic enzyme 2 (ME2) as a candidate susceptibility gene for adolescent-onset GGE. In this article, we present new evidence supporting ME2's involvement in GGE. Methods To definitively test ME2's influence on GGE, we used 3 different approaches. First, we compared a newly recruited GGE cohort with an ethnically matched reference sample from 1000 Genomes Project, using an efficient test of association (POPFAM+). Second, we used POPFAM+ to reanalyze a previously collected data set, wherein the original controls were replaced with ethnically matched reference samples to minimize the confounding effect of population stratification. Third, in a post hoc analysis of expression data from healthy human prefrontal cortex, we identified single nucleotide polymorphisms (SNPs) influencing ME2 messenger RNA (mRNA) expression; and then we tested those same SNPs for association with GGE in a large case-control cohort. Results First, in the analysis of our newly recruited GGE Cohort, we found a strong association between an ME2 SNP and GGE (P = 0.0006 at rs608781). Second, in the reanalysis of previously collected data, we confirmed the Greenberg et al (2005) finding of a GGE-associated ME2 risk haplotype. Third, in the post hoc ME2 expression analysis, we found evidence for a possible link between GGE and ME2 gene expression in human brain. Significance Overall, our research, and the research of others, provides compelling evidence that ME2 influences susceptibility to adolescent-onset GGE.

A biosensor for malic acid detection in fruit juices, based on malate dehydrogenase (MDH) glutamate oxaloacetate transaminase (GOT) in a powder vitreous carbon, carbon nanotubes and mineral oil, composite matrix has been developed. By employing amperometry at a constant potential of 0.4 V (vs. Ag/AgCl), an enzymatic reaction promoted by these species in a support solution Mc Ilvaine buffer (phosphate/citrate pH 10.0) with glutamic acid was generated, causing the oxidation of electro active species, that was stoichiometrically equal to the amount of malic acid present in the sample. The response was linear over the range 2–50 mM, with an analytical sensitivity of 2.24×10−4 to 2,55×10−4 AM−1 and r2=0.9980 to 0.9983, a LOD of 4.02 μM and LOQ of 13.4 μM. Malic acid concentration detected was 0.23–1.07 g/L in fruit juice samples. Results were validated using an enzymatic spectrophotometric method as a reference. To the best of our knowledge, this is the first bienzymatic biosensor using both malate dehydrogenase and glutamate oxaloacetate transaminase enzymes.

Biocatalytic reactions are increasingly being used as a sustainable strategy in organic synthesis and it is recognised that there is need for new enzyme discovery. To establish the utility and versatility of a metagenomics approach, metagenomic DNA extracted from the oral cavity was sequenced and used to create an in silico contig library. This enables individual open reading frames, operons or all the enzymes of a particular family to be identified and then retrieved from the original DNA by PCR. As proof of principle a lactate dehydrogenase, a malate dehydrogenase and transketolases were identified in silico, successfully cloned and assayed. This new enzyme retrieval sequence directed method gives constructive access to metagenomic diversity and importantly improves on the low hit rate experienced when using conventional metagenomic screens.

Cascade reactions drive and regulate a variety of metabolic activities. Efficient coupling of substrate transport between enzymes is important for overall pathway activity and also controls the depletion of intermediate molecules that drive the reaction forward. Here, we assembled a three-enzyme pathway on a series of DNA nanoscaffolds to investigate the dependence of their activities on spatial arrangement. Unlike previous studies, the overall activity of the three-enzyme pathway relied less on inter-enzyme distance and more on the geometric patterns that arranged them within a relatively small range of 10-30 nm. Pathway intermediate detection demonstrated that the assembled enzyme systems quickly depleted the intermediate molecules through efficient reaction coupling.

To determine the effects of docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) ratios on grass carp, Ctenopharyngodon idellus, a 38-d feeding trial was conducted using six isonitrogenous and isoenergetic semi-purified diets containing constant n-3 long-chain polyunsaturated fatty acid (LC-PUFA) (0.5% of dry matter), but varying ratios of DHA to EPA and a control diet (no n-3 LC-PUFA was included). The results revealed higher final weight and specific growth rate in the DHA/EPA 0.21 group. The n-3 LC-PUFA content increased in the CK (control) groups compared with that in the control diet. Lipoprotein lipase (LPL) activity increased in the treatment groups. Malate dehydrogenase showed lower activity in the DHA/EPA 1.08 group, as well as to the change in the level of glucose-6-phosphate dehydrogenase (G6PDH). The gene expressions of LPL increased in the treatment groups and that of peroxisome proliferator-activated receptor α gene showed higher expressions in DHA/EPA 1.08, 0.49, and 0.21 groups. However, no remarkable differences were found among the six groups in the peroxisome proliferator-activated receptor γ gene expression. Our findings indicated that dietary n-3 LC-PUFA affected fatty acid composition and lipid metabolism of grass carp. Further, fish achieved the best effect in decreasing the lipid accumulation when dietary DHA/EPA ratio was not greater than 1.

Purpose Depression is a major disabling psychiatric disorder which causes severe financial burden and social consequences worldwide. Recently, (2R, 6R)-hydroxynorketamine (HNK), a metabolite of ketamine, showed strong antidepressant effect through N-methyl-D-aspartate (NMDA) antagonizing independent mechanism. In the current study the goal is to identify the potential intracellular molecules and pathways that might be involved in different therapeutic effects underlying HNK as compared to NMDA antagonist MK-801. Experimental design Forced-swim behavioral test, 2D fluorescence difference gel electrophoresis, and MALDI-TOF-MS/MS proteomics are used. Results Compared to saline group, 14 differential proteins are identified in MK-801 treated group, with six proteins significantly up-regulated, while in HNK treated group 18 distinct proteins are identified with 11 proteins significantly up-regulated. Likewise, two proteins are significantly upregulated in HNK treated group when compared to MK-801 treated group. Among these differentially expressed proteins, phosphoglycerate mutase 1, malate dehydrogenase/ cytoplasmic, and triosephosphate isomerase are co-affected by MK-801 and HNK treatment. Representative protein expression changes are quantified by western blot, showing consistent results as determined by MALDI-TOF-MS/MS. Conclusion and clinical relevance The core protection mechanisms of HNK observed herein involves improving the abnormal ATP synthesis, impaired glycolysis, and the defense system therefore provides mechanistic insight and molecular targets for novel antidepressants.