A universal metabolite repair enzyme removes a strong inhibitor of the TCA cycle.

A prevalent side-reaction of succinate dehydrogenase oxidizes malate to enol-oxaloacetate (OAA), a metabolically inactive form of OAA that is a strong inhibitor of succinate dehydrogenase. We purified from cow heart mitochondria an enzyme (OAT1) with OAA tautomerase (OAT) activity that converts enol-OAA to the physiological keto-OAA form, and determined that it belongs to the highly conserved and previously uncharacterized Fumarylacetoacetate_hydrolase_domain-containing protein family. From all three domains of life, heterologously expressed proteins were shown to have strong OAT activity, and ablating the OAT1 homolog caused significant growth defects. In Escherichia coli, expression of succinate dehydrogenase was necessary for OAT1-associated growth defects to occur, and ablating OAT1 caused a significant increase in acetate and other metabolites associated with anaerobic respiration. OAT1 increased the succinate dehydrogenase reaction rate by 35% in in vitro assays with physiological concentrations of both succinate and malate. Our results suggest that OAT1 is a universal metabolite repair enzyme that is required to maximize aerobic respiration efficiency by preventing succinate dehydrogenase inhibition.

Effects of glutamate oxaloacetate transaminase on reactive oxygen species in Ganoderma lucidum.

Nitrogen metabolism can regulate mycelial growth and secondary metabolism in Ganoderma lucidum. As an important enzyme in intracellular amino acid metabolism, glutamate oxaloacetate transaminase (GOT) has many physiological functions in animals and plants, but its function in fungi has been less studied. In the present study, two GOT isoenzymes were found in G. lucidum; one is located in the mitochondria (GOT1), and the other is located in the cytoplasm (GOT2). The reactive oxygen species (ROS) level was increased in got1 silenced strains and was approximately 1.5-fold higher than that in the wild-type (WT) strain, while silencing got2 did not affect the ROS level. To explore how GOT affects ROS in G. lucidum, experiments related to the generation and elimination of intracellular ROS were conducted. First, compared with that in the WT strain, the glutamate content, one of the substrates of GOT, decreased when got1 or got2 was knocked down, and the glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH) content decreased by approximately 38.6%, 19.3%, and 40.1% in got1 silenced strains, got2 silenced strains, and got1/2 co-silenced strains respectively. Second, GOT also affects glucose metabolism. The pyruvate (PA), acetyl-CoA and α-ketoglutarate (α-KG) contents decreased in got1 and got2 silenced strains, and the transcription levels of most genes involved in the glycolytic pathway and the tricarboxylic acid cycle increased. The NADH content was increased in got1 silenced strains and got2 silenced strains, and the NAD+/NADH ratio was decreased, which might result in mitochondrial ROS production. Compared with the WT strain, the mitochondrial ROS level was approximately 1.5-fold higher in the got1 silenced strains. In addition, silencing of got1 or got2 resulted in a decrease in antioxidant enzymes, including superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase. Finally, ganoderic acid (GA) was increased by approximately 40% in got1 silenced strains compared with the WT strain, while silencing of got2 resulted in a 10% increase in GA biosynthesis. These findings provide new insights into the effect of GOT on ROS and secondary metabolism in fungi. KEY POINTS: • GOT plays important roles in ROS level in Ganoderma lucidum. • Silencing of got1 resulted in decrease in GSH content and antioxidant enzymes activities, but an increase in mitochondrial ROS level in G. lucidum. • Silencing of got1 and got2 resulted in an increase in ganoderic acid biosynthesis in G. lucidum.

Metabolic adaptation and ATP homeostasis in Pseudomonas fluorescens exposed to phosphate stress.

Phosphate (Pi) is essential for life as it is an integral part of the universal chemical energy adenosine triphosphate (ATP), and macromolecules such as, DNA, RNA proteins and lipids. Despite the core roles and the need of this nutrient in living cells, some bacteria can grow in environments that are poor in Pi. The metabolic mechanisms that enable bacteria to proliferate in a low phosphate environment are not fully understood. In this study, the soil microbe Pseudomonas (P.) fluorescens was cultured in a control and a low Pi (stress) medium in order to delineate how energy homeostasis is maintained. Although there was no significant variation in biomass yield in these cultures, metabolites like isocitrate, oxaloacetate, pyruvate and phosphoenolpyruvate (PEP) were markedly increased in the phosphate-starved condition. Components of the glycolytic, glyoxylate and tricarboxylic acid cycles operated in tandem to generate ATP by substrate level phosphorylation (SLP) as NADH-producing enzymes were impeded. The α-ketoglutarate (KG) produced when glutamine, the sole carbon nutrient was transformed into phosphoenol pyruvate (PEP) and succinyl-CoA (SC), two high energy moieties. The metabolic reprogramming orchestrated by isocitrate lyase (ICL), phosphoenolpyruvate synthase (PEPS), pyruvate phosphate dikinase (PPDK), and succinyl-CoA synthetase fulfilled the ATP budget. Cell free extract experiments confirmed ATP synthesis in the presence of such substrates as PEP, oxaloacetate and isocitrate respectively. Gene expression profiling revealed elevated transcripts associated with numerous enzymes including ICL, PEPS, and succinyl-CoA synthetase (SCS). This microbial adaptation will be critical in promoting biological activity in Pi-poor ecosystems.

Nutritional Component Analyses in Different Varieties of Actinidia eriantha Kiwifruit by Transcriptomic and Metabolomic Approaches.

Actinidia eriantha is a unique germplasm resource for kiwifruit breeding. Genetic diversity and nutrient content need to be evaluated prior to breeding. In this study, we looked at the metabolites of three elite A. eriantha varieties (MM-11, MM-13 and MM-16) selected from natural individuals by using a UPLC-MS/MS-based metabolomics approach and transcriptome, with a total of 417 metabolites identified. The biosynthesis and metabolism of phenolic acid, flavonoids, sugars, organic acid and AsA in A. eriantha fruit were further analyzed. The phenolic compounds accounted for 32.37% of the total metabolites, including 48 phenolic acids, 60 flavonoids, 7 tannins and 20 lignans and coumarins. Correlation analysis of metabolites and transcripts showed PAL (DTZ79_15g06470), 4CL (DTZ79_26g05660 and DTZ79_29g0271), CAD (DTZ79_06g11810), COMT (DTZ79_14g02670) and FLS (DTZ79_23g14660) correlated with polyphenols. There are twenty-three metabolites belonging to sugars, the majority being sucrose, glucose arabinose and melibiose. The starch biosynthesis-related genes (AeglgC, AeglgA and AeGEB1) were expressed at lower levels compared with metabolism-related genes (AeamyA and AeamyB) in three mature fruits of three varieties, indicating that starch was converted to soluble sugar during fruit maturation, and the expression level of SUS (DTZ79_23g00730) and TPS (DTZ79_18g05470) was correlated with trehalose 6-phosphate. The main organic acids in A. eriantha fruit are citric acid, quinic acid, succinic acid and D-xylonic acid. Correlation analysis of metabolites and transcripts showed ACO (DTZ79_17g07470) was highly correlated with citric acid, CS (DTZ79_17g00890) with oxaloacetic acid, and MDH1 (DTZ79_23g14440) with malic acid. Based on the gene expression, the metabolism of AsA acid was primarily through the L-galactose pathway, and the expression level of GMP (DTZ79_24g08440) and MDHAR (DTZ79_27g01630) highly correlated with L-Ascorbic acid. Our study provides additional evidence for the correlation between the genes and metabolites involved in phenolic acid, flavonoids, sugars, organic acid and AsA synthesis and will help to accelerate the kiwifruit molecular breeding approaches.

Targeted metabolomics combined with network pharmacology to reveal the protective role of luteolin in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease that significantly endangers human health, where metabolism may drive pathogenesis: a shift from mitochondrial oxidation to glycolysis occurs in diseased pulmonary vessels and the right ventricle. An increase in pulmonary vascular resistance in patients with heart failure with a preserved ejection fraction portends a poor prognosis. Luteolin exists in numerous foods and is marketed as a dietary supplement assisting in many disease treatments. However, little is known about the protective effect of luteolin on metabolism disorders in diseased pulmonary vessels. In this study, we found that luteolin apparently reversed the pulmonary vascular remodeling of PAH rats by inhibiting the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). Moreover, network pharmacology and metabolomics results revealed that the arachidonic acid pathway, amino acid pathway and TCA cycle were dysregulated in PAH. A total of 14 differential metabolites were significantly changed during the PAH, including DHA, PGE2, PGD2, LTB4, 12-HETE, 15-HETE, PGF2α, and 8-iso-PGF2α metabolites in the arachidonic acid pathway, and L-asparagine, oxaloacetate, N-acetyl-L-ornithine, butane diacid, ornithine, glutamic acid metabolites in amino acid and TCA pathways. However, treatment with luteolin recovered the LTB4, PGE2, PGD2, 12-HETE, 15-HETE, PGF2α and 8-iso-PGF2α levels close to normal. Meanwhile, we showed that luteolin also downregulated the gene and protein levels of COX 1, 5-LOX, 12-LOX, and 15-LOX in the arachidonic acid pathway. Collectively, this work highlighted the metabolic mechanism of luteolin-protected PAH and showed that luteolin would hold great potential in PAH prevention.

Inhibition of ATP-citrate lyase improves NASH, liver fibrosis, and dyslipidemia.

Elevated liver de novo lipogenesis contributes to non-alcoholic steatohepatitis (NASH) and can be inhibited by targeting acetyl-CoA carboxylase (ACC). However, hypertriglyceridemia limits the use of pharmacological ACC inhibitors as a monotherapy. ATP-citrate lyase (ACLY) generates acetyl-CoA and oxaloacetate from citrate, but whether inhibition is effective for treating NASH is unknown. Here, we characterize a new mouse model that replicates many of the pathological and molecular drivers of NASH and find that genetically inhibiting ACLY in hepatocytes reduces liver malonyl-CoA, oxaloacetate, steatosis, and ballooning as well as blood glucose, triglycerides, and cholesterol. Pharmacological inhibition of ACLY mirrors genetic inhibition but has additional positive effects on hepatic stellate cells, liver inflammation, and fibrosis. Mendelian randomization of human variants that mimic reductions in ACLY also associate with lower circulating triglycerides and biomarkers of NASH. These data indicate that inhibiting liver ACLY may be an effective approach for treatment of NASH and dyslipidemia.

Multiple metabolic pathways fuel the truncated tricarboxylic acid cycle of the prostate to sustain constant citrate production and secretion.

OBJECTIVE: The prostate is metabolically unique: it produces high levels of citrate for secretion via a truncated tricarboxylic acid (TCA) cycle to maintain male fertility. In prostate cancer (PCa), this phenotype is reprogrammed, making it an interesting therapeutic target. However, how the truncated prostate TCA cycle works is still not completely understood. METHODS: We optimized targeted metabolomics in mouse and human organoid models in ex vivo primary culture. We then used stable isotope tracer analyses to identify the pathways that fuel citrate synthesis. RESULTS: First, mouse and human organoids were shown to recapitulate the unique citrate-secretory program of the prostate, thus representing a novel model that reproduces this unusual metabolic profile. Using stable isotope tracer analysis, several key nutrients were shown to allow the completion of the prostate TCA cycle, revealing a much more complex metabolic profile than originally anticipated. Indeed, along with the known pathway of aspartate replenishing oxaloacetate, glutamine was shown to fuel citrate synthesis through both glutaminolysis and reductive carboxylation in a GLS1-dependent manner. In human organoids, aspartate entered the TCA cycle at the malate entry point, upstream of oxaloacetate. Our results demonstrate that the citrate-secretory phenotype of prostate organoids is supported by the known aspartate-oxaloacetate-citrate pathway, but also by at least three additional pathways: glutaminolysis, reductive carboxylation, and aspartate-malate conversion. CONCLUSIONS: Our results add a significant new dimension to the prostate citrate-secretory phenotype, with at least four distinct pathways being involved in citrate synthesis. Better understanding this distinctive citrate metabolic program will have applications in both male fertility as well as in the development of novel targeted anti-metabolic therapies for PCa.

Disruption of a putative mitochondrial oxaloacetate shuttle protein in Aspergillus carbonarius results in secretion of malic acid at the expense of citric acid production.

BACKGROUND: In filamentous fungi, transport of organic acids across the mitochondrial membrane is facilitated by active transport via shuttle proteins. These transporters may transfer different organic acids across the membrane while taking others the opposite direction. In Aspergillus niger, accumulation of malate in the cytosol can trigger production of citric acid via the exchange of malate and citrate across the mitochondrial membrane. Several mitochondrial organic acid transporters were recently studied in A. niger showing their effects on organic acid production. RESULTS: In this work, we studied another citric acid producing fungus, Aspergillus carbonarius, and identified by genome-mining a putative mitochondrial transporter MtpA, which was not previously studied, that might be involved in production of citric acid. This gene named mtpA encoding a putative oxaloacetate transport protein was expressed constitutively in A. carbonarius based on transcription analysis. To study its role in organic acid production, we disrupted the gene and analyzed its effects on production of citric acid and other organic acids, such as malic acid. In total, 6 transformants with gene mtpA disrupted were obtained and they showed secretion of malic acid at the expense of citric acid production. CONCLUSION: A putative oxaloacetate transporter gene which is potentially involved in organic acid production by A. carbonarius was identified and further investigated on its effects on production of citric acid and malic acid. The mtpA knockout strains obtained produced less citric acid and more malic acid than the wild type, in agreement with our original hypothesis. More extensive studies should be conducted in order to further reveal the mechanism of organic acid transport as mediated by the MtpA transporter.

Dimerization misalignment in human glutamate-oxaloacetate transaminase variants is the primary factor for PLP release.

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), plays an essential role in the catalytic mechanism of various proteins, including human glutamate-oxaloacetate transaminase (hGOT1), an important enzyme in amino acid metabolism. A recent molecular and genetic study showed that the E266K, R267H, and P300L substitutions in aspartate aminotransferase, the Arabidopsis analog of hGOT1, genetically suppress a developmentally arrested Arabidopsis RUS mutant. Furthermore, CD analyses suggested that the variants exist as apo proteins and implicated a possible role of PLP in the regulation of PLP homeostasis and metabolic pathways. In this work, we assessed the stability of PLP bound to hGOT1 for the three variant and wildtype (WT) proteins using a combined 6 µs of molecular dynamics (MD) simulation. For the variants and WT in the holo form, the MD simulations reproduced the "closed-open" transition needed for substrate binding. This conformational transition was associated with the rearrangement of the P15-R32 small domain loop providing substrate access to the R387/R293 binding motif. We also showed that formation of the dimer interface is essential for PLP affinity to the active site. The position of PLP in the WT binding site was stabilized by a unique hydrogen bond network of the phosphate binding cup, which placed the cofactor for formation of the covalent Schiff base linkage with K259 for catalysis. The amino acid substitutions at positions 266, 267, and 300 reduced the structural correlation between PLP and the protein active site and/or integrity of the dimer interface. Principal component analysis and energy decomposition clearly suggested dimer misalignment and dissociation for the three variants tested in our work. The low affinity of PLP in the hGOT1 variants observed in our computational work provided structural rationale for the possible role of vitamin B6 in regulating metabolic pathways.

High activity and stability of codon-optimized phosphoenolpyruvate carboxylase from Photobacterium profundum SS9 at low temperatures and its application for in vitro production of oxaloacetate.

Phosphoenolpyruvate carboxylase (PEPC) of Photobacterium profundum SS9 can be expressed and purified using the Escherichia coli expression system. In this study, a codon-optimized PEPC gene (OPPP) was used to increase expression levels. We confirmed OPPP expression and purified it from extracts of recombinant E. coli SGJS117 harboring the OPPP gene. The purified OPPP showed a specific activity value of 80.3 U/mg protein. The OPPP was stable under low temperature (5-30 °C) and weakly basic conditions (pH 8.5-10). The enzymatic ability of OPPP was investigated for in vitro production of oxaloacetate using phosphoenolpyruvate (PEP) and bicarbonate. Only samples containing the OPPP, PEP, and bicarbonate resulted in oxaloacetate production. OPPP production system using E. coli could be a platform technology to produce high yields of heterogeneous gene and provide the PEPC enzyme, which has high enzyme activity.

[Novel NADPH-dependent L-aspartate dehydrogenases from the mesophilic nitrogen-fixing bacteria Rhodopseudomonas palustris and Bradyrhizobium japonicum].

The genes encoding putative L-aspartate dehydrogenases (EC 1.4.1.21, ADH) from the mesophilic nitrogen-fixing bacteria Rhodopseudomonas palustris and Bradyrhizobium japonicum were cloned and expressed in Escherichia coli. The respective enzymes in the form of hybrid proteins with N-terminal hexahistidine tags were purified to apparent homogeneity. Both enzymes catalyzed in vitro the reductive amination of oxaloacetate to L-aspartate by an order faster than the reverse reaction at a respective pH optimum of 8.0-9.0 and 9.8; also, the enzymes only catalyzed amination under physiological conditions (pH 7.0-8.0). Their specificity to NADPH was higher by 1-2 orders of magnitude than that to NADH. The apparent KM values of ADHs from R. palustris for oxaloacetate, ammonium, and NADPH at pH 9.0 were 9.2, 11.3, and 0.21 mM, respectively, and the corresponding KM values of ADH from B. japonicum were 21, 4.3, and 0.032 mM, respectively. The amination activity of novel ADHs may be important for the fixation of inorganic nitrogen in vivo and used for the construction of a bacterial strain-producer of L-aspartate by metabolic engineering methods.

Cell wall-associated malate dehydrogenase activity from maize roots.

Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn(2+) and Cu(2+) in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn(2+) and Cu(2+) in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.

Unravelling the gallic acid degradation pathway in bacteria: the gal cluster from Pseudomonas putida.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is widely distributed in nature, being a major phenolic pollutant and a commonly used antioxidant and building-block for drug development. We have characterized the first complete cluster (gal genes) responsible for growth in GA in a derivative of the model bacterium Pseudomonas putida KT2440. GalT mediates specific GA uptake and chemotaxis, and highlights the critical role of GA transport in bacterial adaptation to GA consumption. The proposed GA degradation via the central intermediate 4-oxalomesaconic acid (OMA) was revisited and all enzymes involved have been identified. Thus, GalD is the prototype of a new subfamily of isomerases that catalyses a biochemical step that remained unknown, i.e. the tautomerization of the OMAketo generated by the GalA dioxygenase to OMAenol. GalB is the founding member of a new family of zinc-containing hydratases that converts OMAenol into 4-carboxy-4-hydroxy-2-oxoadipic acid (CHA). galC encodes the aldolase catalysing CHA cleavage to pyruvic and oxaloacetic acids. The presence of homologous gal clusters outside the Pseudomonas genus sheds light on the evolution and ecology of the gal genes in GA degraders. The gal genes were used for expanding the metabolic abilities of heterologous hosts towards GA degradation, and for engineering a GA cellular biosensor.

Molecular characterization and significance of phosphoenolpyruvate carboxykinase in a ruminal bacterium, Streptococcus bovis.

To gain knowledge about the significance of phosphoenolpyruvate (PEP) carboxykinase (PCK) in Streptococcus bovis, the sequence of the gene encoding PCK (pck) was determined. Transcriptional analysis indicated that the pck is transcribed in a monocistronic fashion. The level of pck-mRNA was higher when cells were grown on lactose than on glucose, suggesting that PCK synthesis increases when the growth rate is low. The pck-mRNA level was higher in a mutant lacking ccpA, which encodes the catabolite control protein A (CcpA), than in the parent strain, suggesting that pck transcription is suppressed by CcpA. S. bovis PCK showed oxaloacetate (OAA)-decarboxylating activity, but no PEP-carboxylating activity (reverse reaction). In S. bovis, OAA was speculated to be produced from PEP via pyruvate. Disruption of pck in S. bovis resulted in decreased growth rate and cell yield. When a pck-disrupted mutant was grown in a medium lacking amino acids, the lag phase was longer and the cell yield was lower than the case of the parent strain. These results suggest that pck is involved in the initiation of growth, including the induction of amino acid synthesis and energy metabolism.

Oxaloacetate supplementation increases lifespan in Caenorhabditis elegans through an AMPK/FOXO-dependent pathway.

Reduced dietary intake increases lifespan in a wide variety of organisms. It also retards disease progression. We tested whether dietary supplementation of citric acid cycle metabolites could mimic this lifespan effect. We report that oxaloacetate supplementation increased lifespan in Caenorhabditis elegans. The increase was dependent on the transcription factor, FOXO/DAF-16, and the energy sensor, AMP-activated protein kinase, indicating involvement of a pathway that is also required for lifespan extension through dietary restriction. These results demonstrate that supplementation of the citric acid cycle metabolite, oxaloacetate, influences a longevity pathway, and suggest a tractable means of introducing the health-related benefits of dietary restriction.

Description of Oxalicibacterium horti sp. nov. and Oxalicibacterium faecigallinarum sp. nov., new aerobic, yellow-pigmented, oxalotrophic bacteria.

Three strains of aerobic, Gram-negative, rod-shaped, non-spore-forming, yellow-pigmented bacteria (OD1(T), YOx(T) and NS13), which were isolated in previous studies by enrichment in a mineral medium with potassium oxalate as the sole carbon source, were characterized. On the basis of 16S rRNA gene sequence similarity, strains OD1(T), YOx(T) and NS13 belong to the Betaproteobacteria, most closely related to Oxalicibacterium flavum TA17(T) (97.2-99.7% sequence similarity). The major whole-cell fatty acids were C(16:0), C(16:1)omega7c and C(17:0) cyclo. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains OD1(T) and YOx(T) from O. flavum TA17(T) and from each other. Therefore, it is concluded that the strains OD1(T) and YOx(T) represent novel species within the genus Oxalicibacterium, for which the names Oxalicibacterium horti sp. nov. (type strain OD1(T)=DSM 21640(T)=NBRC 13594(T)) and Oxalicibacterium faecigallinarum sp. nov. (type strain YOx(T)=DSM 21641(T)=CCM 2767(T)) are proposed.

Ethanol extract of Allium sativum attenuates testicular and liver toxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats.

The present study was carried out to investigate the protective role of garlic (Allium sativum) ethanol extract (GE) in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hepatic and testicular toxicity. A total of 60 male rats (Sprague-Dawley, weighing 200 +/- 10 g) were divided into six equal groups. The normal control group (NC) received vehicle (intraperitoneally) and saline (perorally). A predetermined dosage of TCDD (40 microg/kg of body weight, i.p.) was administered to single TCDD-treated (TT) and test (GE) groups. GE was administered (perorally) at daily doses of 5 (GE5), 10 (GE10), 20 (GE 20), or 40 (GE40) mg/kg of body weight for 5 weeks, starting 1 week before the TCDD exposure. Decreases in body weight gain (P < .01) and testicular weight (P < .01) induced by TCDD were greatly attenuated by GE (P < .05-.01). TCDD-induced decreases in spermatogenesis-related panels--Johnsen's score, seminiferous tubular size, ratio of tubules with sperm, and sperm count/tubule--were greatly improved by GE treatment in a dose-dependent manner in the rats. TCDD-induced increases in serum cholesterol and triglyceride levels and glutamic oxaloacetate activity were also suppressed by GE (P < .05-.01). These results indicate that administration of garlic to TCDD-exposed rats attenuates testicular and hepatic damage, suggesting that garlic might be a useful agent that can protect human health from toxic responses induced by environmental pollutants.

Functional evaluation of serine 252 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase mutant Ser252Ala, affecting the conserved Walker A serine residue, was characterized to elucidate the role of this serine residue. The substitution did not result in changes in the protein structure, as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analysis of the mutated enzyme in both directions of the main reaction and in the two secondary reactions showed an approximately 50-fold increase in apparent K(m) for oxaloacetate with minor alterations in the other kinetic parameters. These results show that the hydroxyl group of serine 252 is required for proper oxaloacetate interaction.

Structures of rat cytosolic PEPCK: insight into the mechanism of phosphorylation and decarboxylation of oxaloacetic acid.

The structures of the rat cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK) reported in the PEPCK-Mn2+, -Mn2+-oxaloacetic acid (OAA), -Mn2+-OAA-Mn2+-guanosine-5'-diphosphate (GDP), and -Mn2+-Mn2+-guanosine-5'-tri-phosphate (GTP) complexes provide insight into the mechanism of phosphoryl transfer and decarboxylation mediated by this enzyme. OAA is observed to bind in a number of different orientations coordinating directly to the active site metal. The Mn2+-OAA and Mn2+-OAA-Mn2+GDP structures illustrate inner-sphere coordination of OAA to the manganese ion through the displacement of two of the three water molecules coordinated to the metal in the holo-enzyme by the C3 and C4 carbonyl oxygens. In the PEPCK-Mn2+-OAA complex, an alternate bound conformation of OAA is present. In this conformation, in addition to the previous interactions, the C1 carboxylate is directly coordinated to the active site Mn2+, displacing all of the waters coordinated to the metal in the holo-enzyme. In the PEPCK-Mn2+-GTP structure, the same water molecule displaced by the C1 carboxylate of OAA is displaced by one of the gamma-phosphate oxygens of the triphosphate nucleotide. The structures are consistent with a mechanism of direct in-line phosphoryl transfer, supported by the observed stereochemistry of the reaction. In the catalytically competent binding mode, the C1 carboxylate of OAA is sandwiched between R87 and R405 in an environment that would serve to facilitate decarboxylation. In the reverse reaction, these two arginines would form the CO2 binding site. Comparison of the Mn2+-OAA-Mn2+GDP and Mn2+-Mn2+GTP structures illustrates a marked difference in the bound conformations of the nucleotide substrates in which the GTP nucleotide is bound in a high-energy state resulting from the eclipsing of all three of the phosphoryl groups along the triphosphate chain. This contrasts a previously determined structure of PEPCK in complex with a triphosphate nucleotide analogue in which the analogue mirrors the conformation of GDP as opposed to GTP. Last, the structures illustrate a correlation between conformational changes in the P-loop, the nucleotide binding site, and the active site lid that are important for catalysis.

How does an enzyme recognize CO2?

Phosphoenolpyruvate carboxykinase (PCK) reversibly catalyzes the carboxylation of phosphoenolpyruvate to oxaloacetate. Carbon dioxide, and not bicarbonate ion, is the substrate utilized. Assays of the carboxylation reaction show that initial velocities are 7.6-fold higher when CO(2) is used instead of HCO(3)(-). Two Escherichia coli PCK-CO(2) crystal structures are presented here. The location of CO(2) is the same for both structures; however the orientation of CO(2) is significantly different, likely from the presence of a manganese ion in one of the structures. PCK and the other three known protein-CO(2) crystal structure complexes have been compared; all have CO(2) hydrogen bonding with a basic amino acid side chain (Arg65 or Lys213 in PCK), likely to polarize CO(2) to make the central carbon atom more electrophilic and thus more reactive. Kinetic studies found that the PCK mutant Arg65Gln increased the K(M) for substrates PEP and oxaloacetate but not for CO(2). The unchanged K(M) for CO(2) can be explained since the Arg65Gln mutant likely maintains a hydrogen bond to one of the oxygen atoms of carbon dioxide.