Two-center mechanism for the oxidative decarboxylation of pyruvate by the pyruvate decarboxylating component of the pyruvate dehydrogenase complex of pigeon breast muscle.
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Animals↗
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Results for “Pyruvate Decarboxylase”
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Identification of an AMP-activatable pyruvate dehydrogenase isozyme in embryos and tumors.
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Animals↗
A common structural motif in thiamin pyrophosphate-binding enzymes.
The amino acid sequences of a wide range of enzymes that utilize thiamin pyrophosphate (TPP) as cofactor have been compared. A common sequence motif approximately 30 residues in length was detected, beginning with the highly conserved sequence -GDG- and concluding with the highly conserved sequence -NN-. Secondary structure predictions suggest that the motif may adopt a beta alpha beta fold. The same motif was recognised in the primary structure of a protein deduced from the DNA sequence of a hitherto unassigned open reading frame of Rhodobacter capsulata. This putative protein exhibits additional homology with some but not all of the TPP-binding enzymes.
Amino Acid Sequence↗
Cholinergic system and memory in the rat: effects of chronic ethanol, embryonic basal forebrain brain transplants and excitotoxic lesions of cholinergic basal forebrain projection system.
Oral administration of ethanol (20% v/v) to male Sprague-Dawley rats for different periods of time up to 28 weeks resulted in profound reductions of acetylcholine content, in vitro synthesis and release of acetylcholine, choline uptake, activities of choline acetyltransferase, acetylcholinesterase and pyruvate decarboxylase, content of noradrenaline, serotonin and, to a lesser extent, dopamine throughout the brain. Changes were fully and partially reversible by a 4 weeks' ethanol-free period following a treatment of 8 and 18 weeks, respectively. They remained persistent, however, after 28 weeks of treatment. Performance in an eight arm-radial maze revealed a severe impairment in both spatial and non-spatial reference and working memory. A similar pattern of memory impairment was obtained after ibotenate lesion of the cholinergic basal forebrain projection system. In order to test whether this memory impairment depends on cholinergic deafferentation of the cortex, cholinergic-rich fetal basal forebrain cell suspensions were transplanted into cortex, hippocampus or both these sites in ethanol treated rats. Cholinergic-rich transplants, but not cholinergic-poor transplants, were effective in ameliorating impaired memory function and measures of cholinergic activity in the basal forebrain projection system. The behavioural efficacy of the basal forebrain grafts was well correlated with measures of both transplant volume and the degree to which they restored acetylcholine content at the transplant site; these transplants had no effect, however, on brain monoamine levels. The effects of the cholinergic-rich transplants into cortical and hippocampal sites were additive in their amelioration of performance in the radial maze. Similarly, ibotenate lesions of the sites of origin of the cholinergic projections to neocortex (in the region of the nucleus basalis magnocellularis) and hippocampus (the medial septal areas and nucleus of the diagonal band), respectively, were additive in their deleterious effects on maze performance. There were no qualitative differences in the susceptibility of the four different types of memory performance measured (spatial and non-spatial reference and working memory) to the effects of ethanol, ibotenate lesions of the cholinergic projection system, or cholinergic-rich brain tissue transplants. Thus, overall, the results indicate that the forebrain cholinergic system acts as a whole, without major functional differences between the projections originating in the medial septal area/diagonal band complex and the basal nucleus, and that it discharges a very general function in cognitive processes.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Nonviability of cells with oxidative defects in galactose medium: a screening test for affected patient fibroblasts.
Diagnosis of respiratory chain defects in cultured skin fibroblasts is a difficult diagnostic procedure. We investigated the feasibility of using survival of skin fibroblasts in culture medium with galactose as the major carbon source as a method of quickly diagnosing cell lines that were compromised in oxidative metabolism. We found that cells from patients with most forms of cytochrome oxidase deficiency, cells with complex I deficiency, cells with multiple respiratory chain defects and cells with severe pyruvate dehydrogenase (PDH) complex deficiency failed to survive when subcultured into galactose (5 mM) medium. Cells from patients with Lebers hereditary optic neuropathy (LHON), Kearns-Sayre syndrome (KSS), myoclonus-epilepsy-lactic acidosis-stroke (MELAS), the hepatic form of cytochrome oxidase deficiency, and mild PDH complex deficiency survived well in galactose (5 mM)-containing medium. This could be used as a rapid screening test for skin fibroblasts with major oxidative defects.
Cell Line↗
X-chromosome localization of the functional gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex.
The functional gene locus for the E1 alpha subunit of the human pyruvate dehydrogenase complex has been localized to the p22.1-22.2 region of the X chromosome by in situ hybridization and analysis of somatic cell hybrids with various human X-chromosome rearrangements. Another locus showing significant cross-hybridization with an E1 alpha cDNA probe was detected on chromosome 4, in the region q22. The X-chromosome localization of the pyruvate dehydrogenase E1 alpha subunit gene provides a number of possible explanations for the clinical and biochemical variability which is a major feature of human pyruvate dehydrogenase deficiency.
Animals↗
In silico insight into two rice chromosomal regions associated with submergence tolerance and resistance to bacterial leaf blight and gall midge.
Plants respond to both biotic and abiotic stresses through a common signaling system to provide defense and protection against many adverse environments. Many genes/QTLs governing resistance to both biotic and abiotic stresses have been studied and mapped in rice. Sub1, a major QTL for submergence tolerance is collocated with a gene Gm1 for gall midge resistance on chromosome 9 (Region 1). Likewise a bigger region on chromosome 5 (Region 2) has a minor QTL for submergence tolerance collocated with genes for bacterial blight resistance. Utilizing the rice sequence and annotation data (TIGR) and rice genome annotation project database (RAP-DB), we wanted to know the kinds of genes underlying these two chromosomal regions where genes/QTL governing tolerance to both biotic and abiotic stresses are collocated. We also analyzed the pattern of distribution of these genes across the BAC/PAC clones spanning the region so that candidate genes can be short listed for a functional analysis. Genes known to have a role in submergence tolerance were present in both the regions. Region 1, had a unique transcription factor like trithorax protein, which is a positional candidate gene for submergence tolerance. Pyruvate decarboxylase (PDC) gene for alcohol fermentation and cation transporting ATPase c-terminal domain are likely candidates for submergence QTL in Region 2. Genes such as SKP1 and elicitor induced cytochrome p450 associated with tissue necrosis and insect resistance were found in region 1. Multiple copies of ORFs for signal transduction proteins, transcription factors, genes for systemic acquired resistance, Ubiquitin proteins and pathogen elicitor identification and degrading proteins were located as a cluster in Region 2, where bacterial blight resistance genes mapped. Validation of the data obtained from TIGR with other databases (RAP and KOME) confirmed our findings. The functional role of some of the significant candidate genes needs to be established. Allele/gene specific markers can then be designed for use in MAS thus enhancing durable tolerance/resistance faster.
Adaptation, Physiological↗
Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled.
Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38 degrees C in a standard minimal medium (MMAB) containing 2.5 gl(-1)l-malate and 50 microgml(-1) tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene (ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense.
Aerobiosis↗
Yeast glycolytic oscillations that are not controlled by a single oscillophore: a new definition of oscillophore strength.
Biochemical oscillations, such as glycolytic oscillations, are often believed to be caused by a single so-called 'oscillophore'. The main characteristics of yeast glycolytic oscillations, such as frequency and amplitude, are however controlled by several enzymes. In this paper, we develop a method to quantify to which extent any enzyme determines the occurrence of oscillations. Principles extrapolated from metabolic control analysis are applied to calculate the control exerted by individual enzymes on the real and imaginary parts of the eigenvalues of the Jacobian matrix. We propose that the control exerted by an enzyme on the real part of the smallest eigenvalue, in terms of absolute value, quantifies to which extent that enzyme contributes to the emergence of instability. Likewise the control exerted by an enzyme on the imaginary part of complex eigenvalues may serve to quantify the extent to which that enzyme contributes to the tendency of the system to oscillate. The method was applied both to a core model and to a realistic model of yeast glycolytic oscillations. Both the control over stability and the control over oscillatory tendency were distributed among several enzymes, of which glucose transport, pyruvate decarboxylase and ATP utilization were the most important. The distributions of control were different for stability and oscillatory tendency, showing that control of instability does not imply control of oscillatory tendency nor vice versa. The control coefficients summed up to 1, suggesting the existence of a new summation theorem. These results constitute proof that glycolytic oscillations in yeast are not caused by a single oscillophore and provide a new, subtle, definition for the oscillophore strength of an enzyme.
Biological Clocks↗
Reducing properties, energy efficiency and carbohydrate metabolism in hyperhydric and normal carnation shoots cultured in vitro: a hypoxia stress?
Hyperhydricity is considered as a physiological disorder that can be induced by different stressing conditions. In the present work we have studied the metabolic and energetic states of hyperhydric carnation shoots. We have evaluated the hypothesis that hypoxia stress is the main factor affecting the metabolism of hyperhydric leaves. Our results indicate a low level of ATP in hyperhydric tissues, but only slight modifications in pyridine nucleotide contents. Concurrently, the glucose-6-phosphate dehydrogenase (G-6-PDH; EC 1.1.1.49) activity in hyperhydric leaves was increased but glucokinase (GK; EC 2.7.1.2) activity was unchanged. We have observed that the metabolism of pyruvate was altered in hyperhydric tissues by the induction of pyruvate synthesis via NADP-dependent malic enzyme (EC 1.1.1.40). The enzymes of the fermentative metabolism pyruvate decarboxylase (PDC; EC 4.1.1.1) and alcohol dehydrogenase (ADH; EC 1.1.1.1) were highly increased in hyperhydric leaves. Sucrose metabolism was modified in hyperhydric leaves with a high increase in the activity of both synthesis and catabolic enzymes. The analysis of the sucrose, glucose and fructose contents indicated that all of these sugars were accumulated in hyperhydric leaves. However, the pinitol content was drastically decreased in hyperhydric leaves. We consider that these results suggest that hyperhydric leaves of carnation have adapted to hypoxia stress conditions by the induction of the oxidative pentose phosphate and fermentative pathways.
Carbohydrate Metabolism↗
Engineering of Saccharomyces cerevisiae for the production of L-glycerol 3-phosphate.
L-glycerol 3-phosphate (L-G3P) was accumulated in Saccharomyces cerevisiae by pathway engineering. Intracellular concentration of this metabolic intermediate could be increased more than 20 times compared to the wild type by overexpressing GPD1 encoding the glycerol 3-phosphate dehydrogenase in a gpp1 Delta gpp2 Delta mutant which lacks both isoenzymes of glycerol 3-phosphatase. Investigation of cellular pattern of triacylglycerols and glycerophospholipids did not reveal considerable changes due to accumulation of their precursor L-G3P. Hyperosmotic stress did not affect the L-G3P pool in the gpp1 Delta gpp2 Delta mutant overexpressing GPD1 despite an about 4-fold increase of specific GPD activity. In contrast, oxygen limitation improved intracellular L-G3P concentration by enhancing the availability of cytosolic NADH. The reduction of pyruvate decarboxylase activity by deleting PDC2 led to an additional increase. In fact, the triple mutant gpp1 Delta gpp2 Delta pdc2 Delta overexpressing GPD1 accumulated 17 mg L-G3P/g dry weight during glucose batch fermentation under oxygen limitation. This value corresponds to an about 100-fold increase compared to that found in the wild type.
Genetic Enhancement↗
Physiological and genetic engineering of cytosolic redox metabolism in Saccharomyces cerevisiae for improved glycerol production.
Previous metabolic engineering strategies for improving glycerol production by Saccharomyces cerevisiae were constrained to a maximum theoretical glycerol yield of 1 mol.(molglucose)(-1) due to the introduction of rigid carbon, ATP or redox stoichiometries. In the present study, we sought to circumvent these constraints by (i) maintaining flexibility at fructose-1,6-bisphosphatase and triosephosphate isomerase, while (ii) eliminating reactions that compete with glycerol formation for cytosolic NADH and (iii) enabling oxidative catabolism within the mitochondrial matrix. In aerobic, glucose-grown batch cultures a S. cerevisiae strain, in which the pyruvate decarboxylases the external NADH dehydrogenases and the respiratory chain-linked glycerol-3-phosphate dehydrogenase were deleted for this purpose, produced glycerol at a yield of 0.90 mol.(molglucose)(-1). In aerobic glucose-limited chemostat cultures, the glycerol yield was ca. 25% lower, suggesting the involvement of an alternative glucose-sensitive mechanism for oxidation of cytosolic NADH. Nevertheless, in vivo generation of additional cytosolic NADH by co-feeding of formate to aerobic, glucose-limited chemostat cultures increased the glycerol yield on glucose to 1.08 mol mol(-1). To our knowledge, this is the highest glycerol yield reported for S. cerevisiae.
Bioreactors↗
Mechanism of action of oxfenicine on muscle metabolism.
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Aminooxyacetic Acid↗
Interplay of organic and biological chemistry in understanding coenzyme mechanisms: example of thiamin diphosphate-dependent decarboxylations of 2-oxo acids.
With the publication of the three-dimensional structures of several thiamin diphosphate-dependent enzymes, the chemical mechanism of their non-oxidative and oxidative decarboxylation reactions is better understood. Chemical models for these reactions serve a useful purpose to help evaluate the additional catalytic rate acceleration provided by the protein component. The ability to generate, and spectroscopically observe, the two key zwitterionic intermediates invoked in such reactions allowed progress to be made in elucidating the rates and mechanisms of the elementary steps leading to and from these intermediates. The need remains to develop chemical models, which accurately reflect the enzyme-bound conformation of this coenzyme.
Acetoin Dehydrogenase↗
Influence of yeast immobilization on fermentation and aldehyde reduction during the production of alcohol-free beer.
Production of alcohol-free beer by limited fermentation is optimally performed in a packed-bed reactor. This highly controllable system combines short contact times between yeast and wort with the reduction of off-flavors to concentrations below threshold values. In the present study, the influence of immobilization of yeast to DEAE-cellulose on sugar fermentation and aldehyde reduction was monitored. Immobilized cells showed higher activities of hexokinase and pyruvate decarboxylase compared to cells grown in batch culture. In addition, a higher glucose flux was observed, with enhanced excretion of main fermentation products, indicating a reduction in the flux of sugar used for biomass production. ADH activity was higher in immobilized cells compared to that in suspended cells. However, during prolonged production a decrease was observed in NAD-specific ADH activity, whereas NADP-specific activity increased in the immobilized cells. The shifts in enzyme activities and glucose flux correlate with a higher in vivo reduction capacity of the immobilized cells.
Journal Article↗
Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae.
Regulation of fermentative capacity was studied in chemostat cultures of two Saccharomyces cerevisiae strains: the laboratory strain CEN.PK113-7D and the industrial bakers' yeast strain DS28911. The two strains were cultivated at a fixed dilution rate of 0.10 h(-1) under various nutrient limitation regimes: aerobic and anaerobic glucose limitation, aerobic and anaerobic nitrogen limitation on glucose, and aerobic ethanol limitation. Also the effect of specific growth rate on fermentative capacity was compared in glucose-limited, aerobic cultures grown at dilution rates between 0.05 h(-1) and 0.40 h(-1). Biomass yields and metabolite formation patterns were identical for the two strains under all cultivation conditions tested. However, the way in which environmental conditions affected fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions) differed for the two strains. A different regulation of fermentative capacity in the two strains was also evident from the levels of the glycolytic enzymes, as determined by in vitro enzyme assays. With the exception of phosphofructokinase and pyruvate decarboxylase in the industrial strain, no clear-cut correlation between the activities of glycolytic enzymes and the fermentative capacity was found. These results emphasise the need for controlled cultivation conditions in studies on metabolic regulation in S. cerevisiae and demonstrate that conclusions from physiological studies cannot necessarily be extrapolated from one S. cerevisiae strain to the other.
Journal Article↗
Fermentative capacity after cold storage of baker's yeast is dependent on the initial physiological state but not correlated to the levels of glycolytic enzymes.
Growth and starvation of baker's yeast was monitored by on-line microcalorimetry and cells originating from four different physiological states were stored at low temperature (4 degrees C) for up to 26 days. The different physiological states were designated F (respiro-Fermentative phase of growth), R (initial Respiratory phase of growth), -N (non-growing state because of Nitrogen depletion), and -NC (non-growing state because of both Nitrogen and Carbon depletion). The cells were tested before and after cold storage for their fermentative capacity, and characterised by 2D gel analysis (and subsequent quantitative silver staining and image analysis with software PDQUEST) for their levels of six enzymes of the glycolytic pathway (hexokinase 2 (Hxk2p), fructose bisphosphate aldolase (Fba1p), glyceraldehyde-3-phosphate dehydrogenase (Tdh3p), enolase A (Enolp), enolase B (Eno2p), and triose phosphate isomerase (Tpi1p)) and two enzymes of the fermentative branch (pyruvate decarboxylase (Pdc1p) and alcohol dehydrogenase (Adh1p)). The enzymes Hxk2p, Tdh3p, Eno2p, Pdc1p and Adh1p were down-regulated by 25-80% during the transition between the F and R states. During the transition to non-growing states (-N and -NC states), the levels of Hxk2p, Tdh3p and Eno2p were further reduced. However, after cold storage, the glycolytic and fermentative enzymes of the different physiological states were expressed to the same extent. In contrast, the fermentative capacity differed between the states; the R-state cells were superior compared to cells from the other states tested and preserved more than 50% of their initial fermentative capacity (6 mmol ethanol per gram dry weight and hour). Our data therefore clearly demonstrate that persistence of fermentative capacity during total starvation at low temperature after as long as 1 month is strongly dependent on the physiological state from which the cells originate. However, the level of expression of the glycolytic enzymes could not explain the difference in fermentative capacity of the different physiological states after cold storage.
Calorimetry↗
Cloning and phylogenetic analysis of the genes encoding acetohydroxyacid synthase from the archaeon Methanococcus aeolicus.
The gene for acetohydroxyacid synthase (AHAS) was cloned from the archaeon Methanococcus aeolicus. Contrary to biochemical studies [Xing, R. and Whitman, W.B. (1994) J. Bacteriol. 176, 1207-1213] the enzyme was encoded by two open reading frames (ORFs). Based on sequence homology, these ORFs were designated ilvB and ilvN for the large and small subunits of AHAS, respectively. A putative methanogen promoter preceded ilvB-ilvN, and a potential internal promoter was found upstream of ilvN. ilvB encoded a 65-kDa protein, which agreed well with the measured value for the purified enzyme. ilvN encoded a 19-kDa protein, which fell within the range of M(r) of small subunits from other sources. Phylogenetic analysis of the deduced amino acid sequence of ilvB showed a close relationship between the AHAS of Bacteria and Archaea, to the exclusion of other enzymes in this family, including pyruvate oxidase, glyoxylate carboligase, pyruvate decarboxylase, and the acetolactate synthase found in fermentative Bacteria. Thus, this family of enzymes probably arose prior to the divergence of the Bacteria and Archaea. Moreover, the higher plant AHAS and the red algal AHAS were related to the AHAS II of Escherichia coli and the cyanobacterial AHAS, respectively. For this reason, these genes appear to have been acquired by the Eucarya during the endosymbiosis that gave rise to the mitochondrion and chloroplast, respectively. One of the ORFs in the Methanococcus jannaschii genome possesses high similarity to the M. aeolicus ilvB, indicating that it is an authentic AHAS.
Acetolactate Synthase↗