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PubMed2000

Quaternary organization of the Staphylothermus marinus phosphoenolpyruvate synthase: angular reconstitution from cryoelectron micrographs with molecular modeling.

Digital electron images of frozen-hydrated preparations of the 2.25-MDa Staphylothermus marinus phosphoenolpyruvate synthase (EC 2.7.9.2) have been analyzed by single-particle classification and averaging and iterative quaternion-based angular reconstitution. Contrast transfer function correction of micrographs obtained at different defocus values was used to improve the informational quality of the projection averages. Three-dimensional reconstructions were obtained to roughly 3-nm spatial resolution, in which the 24 identical subunits were arranged to form an octahedral complex, although the amino-terminal nucleotide-binding domain was not resolved. An atomic model of the subunit was generated by homology modeling using as the reference the known X-ray crystallographic structure of the related enzyme pyruvate orthophosphate dikinase (EC 2.7.9.1) from Clostridium symbiosum (Protein Data Bank entry 1DIK). The S. marinus protein could be arranged into an assembly of 12 homodimers to match the three-dimensional reconstruction in terms of shape and size of the homodimers, as well as overall shape and size of the complex. The quaternary model indicated that active sites of three monomers were localized around cavities (or putative channels) centered at the threefold axes of rotational symmetry and that carboxyl-terminal alpha-helical segments of four monomers were localized at the fourfold axes of rotational symmetry where they could facilitate interdimer interaction. The quaternary arrangement also indicated numerous potential hydrophobic and electrostatic interactions at the interdimer interfaces that could contribute further to structural stability.

Bacterial Proteins↗
PubMedDate not supplied

Life with carbon monoxide.

This review focuses on how microbes live on CO as a sole source of carbon and energy and with CO by generating carbon monoxide as a metabolic intermediate. The use of CO is a property of organisms that use the Wood-L jungdahl pathway of autotrophic growth. The review discusses when CO metabolism originated, when and how it was discovered, and what properties of CO are ideal for microbial growth. How CO sensing by a heme-containing transcriptional regulatory protein activates the expression of CO metabolism-linked genes is described. Two metalloenzymes are the cornerstones of growth with CO: CO dehydrogenase (CODH) and acetyl-CoA synthase (ACS). CODH oxidizes CO to CO2, providing low-potential electrons for the cell, or alternatively reduces CO2 to CO. The latter reaction, when coupled to ACS, forms a machine for generating acetyl-CoA from CO2 for cell carbon synthesis. The recently solved crystal structures of CODH and ACS along with spectroscopic measurements and computational studies provide insights into novel bio-organometallic catalytic mechanisms and into the nature of a 140 A gas channel that coordinates the generation and utilization of CO. The enzymes that are coupled to CODH/ACS are also described, with a focus on a corrinoid protein, a methyltransferase, and pyruvate ferredoxin oxidoreductase.

Acetate-CoA Ligase↗
PubMed1981

Biosynthetic and bioenergetic functions of citric acid cycle reactions in Rhodopseudomonas capsulata.

Rhodopseudomonas capsulata can grow in a number of alternative modes, including (i) photosynthetic, defined here as anaerobic growth with light as the energy source, and (ii) heterotrophic, referring to aerobic heterotrophic growth in darkness. The functions of citric acid cycle sequences in these growth modes were investigated using wild-type and appropriate mutant strains. Results of growth tests and O(2) utilization experiments showed that in the heterotrophic mode, energy conversion is dependent on operation of the classical citric acid cycle. Alpha-ketoglutarate dehydrogenase (KGD) activity in wild-type strain B10 is substantially higher in cells grown heterotrophically than in cells grown photosynthetically. Molecular oxygen, even at low concentration, appears to be important in regulation of KGD synthesis and, thus, in expression of citric acid cycle activity. Extracts of (photosynthetically grown) mutant strain KGD11 lack demonstrable KGD activity, and in contrast to the wild type, KGD11 is unable to grow heterotrophically on succinate, malate, or pyruvate owing to failure of the energy conversion function of the citric acid cycle. KGD11, however, grows well photosynthetically on malate or on CO(2) + H(2). The KGD activity level required to support the bioenergetic function of the citric acid cycle is evidently much higher than that necessary to satisfy biosynthetic demands; thus, a very low rate of succinyl-coenzyme A formation (needed for biosynthesis) in the mutant would suffice for growth under photosynthetic conditions. In wild-type R. capsulata, the alpha-ketoglutarate required for glutamate synthesis is ordinarily generated via citric acid cycle reactions, which include the conversions catalyzed by citrate synthase and isocitrate dehydrogenase. Mutants blocked in the former or both of these enzymes can grow photosynthetically if provided with an exogenous source of alpha-ketoglutarate or glutamate, but grow very poorly (if at all) as heterotrophs since the energy supply under these conditions depends on operation of the complete citric acid cycle.

Citrate (si)-Synthase↗
PubMed1967

Carnitine and derivatives in rat tissues.

1. Free carnitine, acetylcarnitine, short-chain acylcarnitine and acid-insoluble carnitine (probably long-chain acylcarnitine) have been measured in rat tissues. 2. Starvation caused an increase in the proportion of carnitine that was acetylated in liver and kidney; at least in liver fat-feeding had the same effect, whereas a carbohydrate diet caused a very low acetylcarnitine content. 3. In heart, on the other hand, starvation did not cause an increase in the acetylcarnitine/carnitine ratio, whereas fat-feeding caused a decrease. The acetylcarnitine content of heart was diminished by alloxan-diabetes or a fatty diet, but not by re-feeding with carbohydrate. 4. Under conditions of increased fatty acid supply the acid-insoluble carnitine content was increased in heart, liver and kidney. 5. The acylation state of carnitine was capable of very rapid change. Concentrations of carnitine derivatives varied with different methods of obtaining tissue samples, and very little acid-insoluble carnitine was found in tissues of rats anaesthetized with Nembutal. In liver the acetylcarnitine (and acetyl-CoA) content decreased if freezing of tissue samples was delayed; in heart this caused an increase in acetylcarnitine. 6. Incubation of diaphragms with acetate or dl-beta-hydroxybutyrate caused the acetylcarnitine content to become elevated. 7. Perfusion of hearts with fatty acids containing an even number of carbon atoms, dl-beta-hydroxybutyrate or pyruvate resulted in increased contents of acetylcarnitine and acetyl-CoA. Accumulation of these acetyl compounds was prevented by the additional presence of propionate or pentanoate in the perfusion medium; this prevention was not due to extensive propionylation of CoA or carnitine. 8. Perfusion of hearts with palmitate caused a severalfold increase in the content of acid-insoluble carnitine; this increase did not occur when propionate was also present. 9. Comparison of the acetylation states of carnitine and CoA in perfused hearts suggests that the carnitine acetyltransferase reactants may remain near equilibrium despite wide variations in their steady-state concentrations. This is not the case with the citrate synthase reaction. It is suggested that the carnitine acetyltransferase system buffers the tissue content of acetyl-CoA against rapid changes.

Journal Article↗
PubMed2006

Tetrahydrobiopterin availability in Parkinson's and Alzheimer's disease; potential pathogenic mechanisms.

Within the central nervous system, tetrahydrobiopterin (BH4) is an essential cofactor for dopamine and serotonin synthesis. In addition, BH4 is now established to be an essential cofactor for all isoforms of nitric oxide synthase (NOS). Inborn errors of metabolism affecting BH4 availability are well documented and the clinical presentation can be attributed to a paucity of dopamine, serotonin, and nitric oxide (NO) generation. In this article, we have focussed upon the sensitivity of BH4 to oxidative catabolism and the observation that when BH4 is limiting some cellular sources of NOS may generate superoxide whilst other BH4 saturated NOS enzymes may be generating NO. Such a scenario could favor peroxynitrite generation. If peroxynitrite is not scavenged, e.g., by antioxidants such as reduced glutathione, irreversible damage to critical cellular enzymes could ensue. Such targets include components of the mitochondrial electron transport chain, alpha ketoglutarate dehydrogenase and possibly pyruvate dehydrogenase. Such a cascade of events is hypothesized, in this article, to occur in neurodegenerative conditions such as Parkinson's and Alzheimer's disease.

Alzheimer Disease↗
PubMed2002

Identification, cloning, purification, and enzymatic characterization of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate synthase.

The enzyme encoded by Rv2682c in Mycobacterium tuberculosis is a functional 1-deoxy-D-xylulose 5-phosphate synthase (DXS), suggesting that the pathogen utilizes the mevalonate-independent pathway for isopentenyl diphosphate and subsequent polyprenyl phosphate synthesis. These key precursors are vital in the biosynthesis of many essential aspects of the mycobacterial cell wall. Rv2682c encodes the conserved DRAG sequence that has been proposed as a signature motif for DXSs and also all 13 conserved amino acid residues thought to be important to the function of transketolase enzymes. Recombinant Rv2682c is capable of utilizing glyceraldehyde 3-phosphate and erythrose 4-phosphate as well as D- and L-glyceraldehyde as aldose substrates. The enzyme has K(m) values of 40 microM, 6.1 microM, 5.6 mM, and 4.5 mM for pyruvate, D-glyceraldehyde 3-phosphate, D-glyceraldehyde, and L-glyceradehyde, respectively. Rv2682c has an absolute requirement for divalent cation and thiamin diphosphate as cofactors. The K(d) (thiamin diphosphate )for the native M. tuberculosis DXS activity partially purified from M. tuberculosis cytosol is 1 microM in the presence of Mg(2+).

Amino Acid Sequence↗
PubMed2002

Intermediate trapping via a conformational switch in the Na(+)-activated tryptophan synthase bienzyme complex.

The tryptophan synthase bienzyme complex channels substrate indole between the alpha- and beta-sites via a 25 A long interconnecting tunnel. Channeling efficiency is dependent upon a conformational switch in alphabeta-dimeric units between open conformations of low activity to which substrates bind and closed conformations of high activity wherein substrates react. In experiments designed to gain a better understanding of the linkage between chemical steps and conformational transitions in the catalytic cycle, the novel amino acid dihydroiso-L-tryptophan (DIT) was used as an analogue of L-Trp. In the forward reaction (indoline + L-Ser) to synthesize DIT, the quinonoid species, E(Q)(indoline), is formed quickly, while in the reverse reaction (DIT cleavage), the accumulation of E(Q)(indoline) occurs very slowly. Nevertheless, when the alpha-site substrate analogue alpha-D,L-glycerol phosphate (GP) is bound, DIT cleavage was found to give a rapid formation and dissipation of E(Q)(indoline) followed by a very slow reappearance of E(Q)(indoline). This result led to the conclusion that the reaction of DIT proceeds quickly through the quinonoid state to give indoline and the alpha-aminoacrylate Schiff base, E(A-A), both in the absence and in the presence of GP. In the absence of GP the slow conversion of E(A-A) to pyruvate and ammonium ion limits the rate of accumulation of free indoline and therefore the rate of buildup of E(Q)(indoline). However, when GP is bound to the alpha-site, the indoline generated by DIT cleavage in the first turnover is trapped within the enzyme complex, shifting the equilibrium distribution strongly in favor of E(Q)(indoline) as a consequence of the high local concentration of sequestered indoline. This sequestering is the result of a switching of alphabeta-subunit pairs to a closed conformation when GP binds to the alpha-site and E(A-A) and/or E(Q)(indoline) is formed at the beta-site, thereby trapping indoline inside. The decay of the transiently formed E(Q)(indoline) occurs due to leakage of indoline from the closed system.

Allosteric Regulation↗
PubMed2002

Imazethapyr, an inhibitor of the branched-chain amino acid biosynthesis, induces aerobic fermentation in pea plants.

Acetolactate synthase (ALS; EC 4.1.3.18) inhibition is the primary mechanism of action of imazethapyr (IM). However, the precise mechanisms that links ALS inhibition with plant death have not been elucidated. Supply of IM to pea (Pisum sativum L) plants produced an immediate cessation of growth, caused a 50% inhibition of the in vivo ALS activity within 1 day of treatment, and a remarkable accumulation (2.7-times) of free amino acids after 3 days. Carbohydrates (soluble and starch) were accumulated in both leaves and roots. Accumulation of soluble sugars in roots preceded that of starch in leaves, suggesting that the accumulation of carbohydrates in leaves is not the reason for the arrested root growth. A transient pyruvate accumulation was observed in roots, 1 day after the onset of IM supply. This was coincident with an increase in pyruvate decarboxylase (EC 4.1.1.1), and later increases in alcohol dehydrogenase (EC 1.1.1.1), lactate dehydrogenase (EC 1.1.1.27), and alanine amino transferase (EC 2.6.1.2) activities. This enhancement of fermentative activities was coincident with a slight decrease in aerobic respiration. The overall data suggest that the impairment of ALS activity may lead to a fermentative metabolism that may be involved in growth inhibition and plant death.

Journal Article↗
PubMed1991

Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site.

The active site of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been probed using site-directed mutagenesis and inhibitor binding techniques. Replacement of a specific glycyl with an alanyl or a prolyl with a seryl residue in a highly conserved region confers glyphosate tolerance to several bacterial and plant EPSPS enzymes, suggesting a high degree of structural conservation between these enzymes. The glycine to alanine substitution corresponding to Escherichia coli EPSPS G96A increases the Ki(app) (glyphosate) of petunia EPSPS 5000-fold while increasing the Km(app)(phosphoenolpyruvate) about 40-fold. Substitution of this glycine with serine, however, abolishes EPSPS activity but results in the elicitation of a novel EPSP hydrolase activity whereby EPSP is converted to shikimate 3-phosphate and pyruvate. This highly conserved region is critical for the interaction of the phosphate moiety of phosphoenolpyruvate with EPSPS.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1985

Submitochondrial localization of arginase and other enzymes associated with urea synthesis and nitrogen metabolism, in liver of Squalus acanthias.

The submitochondrial localization of the four mitochondrial enzymes associated with urea synthesis in liver of Squalus acanthias (spiny dogfish), a representative elasmobranch, was determined. Glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, ornithine carbamoyltransferase, glutamine synthetase, and arginase were all localized within the matrix of liver mitochondria. The subcellular and submitochondrial localization and activities of several related enzymes involved in nitrogen metabolism and gluconeogenesis in liver and dogfish are also reported. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase were localized in the mitochondrial matrix. Synthesis of citrulline by isolated mitochondria from ornithine proceeds at a near optimal rate at ornithine concentrations as low as 0.08 mM. The same stoichiometry and rates of citrulline synthesis are observed when ornithine is replaced by arginine. The mitochondrial location of arginase does not appear to reflect a mechanism for regulating ornithine availability.

Animals↗
PubMed2003

Microdialysis-evaluated myocardial cyclooxygenase-mediated inflammation and early circulatory depression in porcine endotoxemia.

OBJECTIVE: To evaluate the early myocardial biochemical inflammatory response with the microdialysis technique during porcine endotoxemia and to simultaneously monitor systemic hemodynamics. DESIGN: Prospective, randomized, placebo-controlled trial with parallel groups. SETTING: Animal research laboratory at the University Hospital of Uppsala, Sweden. SUBJECTS: Thirteen piglets aged 12-14 wks receiving general anesthesia. INTERVENTIONS: After thoracotomy and the insertion of microdialysis probes in standardized locations in the left ventricle of the heart and in the quadriceps muscle, seven pigs received a continuous infusion of endotoxin, initiating a severe endotoxemic shock. Six pigs received saline instead of endotoxin. MEASUREMENTS AND MAIN RESULTS: Endotoxemia caused a rapid and pronounced elevation of a metabolite obtained from prostaglandin degradation, 15-keto-dihydro-PGF(2alpha), in myocardial microdialysate fluid being specific of cyclooxygenase (COX)-mediated inflammation (p <.001 vs. saline-infused controls). Simultaneously, we observed a decrease in left ventricular stroke work index in the endotoxemic pigs (p <.01 vs. saline-infused controls). Endotoxemia did not alter 15-keto-dihydro-PGF(2alpha) levels in quadriceps muscle. Endotoxemia caused increases in taurine, hypoxanthine, and magnesium in myocardial microdialysate (p <.05 vs. saline-infused controls), whereas the contents of pyruvate, lactate, inosine, adenosine, and calcium were not significantly changed. CONCLUSION: Endotoxemia induced a myocardial COX-mediated inflammation without signs of ischemia. In parallel, a depletion of myocardial energy substrates and a deterioration in myocardial performance were seen.

Analysis of Variance↗
PubMed2003

Some practical aspects of providing a diagnostic service for respiratory chain defects.

The oxidative phosphorylation system (OXPHOS) is organized into five multi-protein complexes, comprising four complexes (I-IV) of the respiratory chain and ATP synthase (complex V). OXPHOS has a vital role in cellular energy metabolism and ATP production. Enzyme analysis of individual OXPHOS complexes in a skeletal muscle biopsy remains the mainstay of the diagnostic process for patients suspected of mitochondrial cytopathy. Practical guidelines are presented to provide optimal conditions for performance of laboratory investigations and a reliable diagnosis. A fresh muscle biopsy is preferable to a frozen muscle sample because the overall capacity of the OXPHOS system can be measured in a fresh biopsy. In about 25% of patients referred for muscle biopsy to our centre, reduced substrate oxidation rates and ATP+creatine phosphate production rates were found without any defect in complexes I-V and the pyruvate dehydrogenase complex. Investigation of frozen muscle biopsy alone may lead to false-negative diagnoses in many patients. In some patients, it is necessary to investigate fibroblasts for prospective diagnostic purposes. An exact diagnosis of respiratory chain defects is a prerequisite for rational therapy and genetic counselling. Provided guidelines for specimen collection are followed, there are now reliable methods for identifying respiratory chain defects.

Adenosine Triphosphate↗
PubMed2002

Subcellular localization of the hypusine-containing eukaryotic initiation factor 5A by immunofluorescent staining and green fluorescent protein tagging.

Eukaryotic initiation factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid residue formed posttranslationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Although the eIF-5A gene is essential for cell survival and proliferation, the precise function and localization of eIF-5A remain unclear. In this study, we have determined the subcellular distribution of eIF-5A by indirect immunofluorescent staining and by direct visualization of green fluorescent protein tagged eIF-5A (GFP-eIF5A). Immunofluorescent staining of the formaldehyde-fixed cells showed that eIF-5A was present in both the nucleus and cytoplasm. Only the nuclear eIF-5A was resistant to Triton extraction. Direct visualization of GFP tagged eIF-5A in living cells revealed the same whole-cell distribution pattern. However, a fusion of an additional pyruvate kinase (PK) moiety into GFP-eIF-5A precluded the nuclear localization of GFP-PK-eIF-5A fusion protein. Fusion of the GFP-PK tag with three different domains of eIF-5A also failed to reveal any nuclear localization of the fusion proteins, suggesting the absence of receptor-mediated nuclear import. Using interspecies heterokaryon fusion assay, we could detect the nuclear export of GFP-Rev, but not of GFP-eIF-5A. The whole-cell distribution pattern of eIF-5A was recalcitrant to the treatments that included energy depletion, heat shock, and inhibition of transcription, translation, polyamine synthesis, or CRM1-dependent nuclear export. Collectively, our data indicate that eIF-5A gains nuclear entry via passive diffusion, but it does not undergo active nucleocytoplasmic shuttling.

3T3 Cells↗
PubMed2004

Identification of S-nitrosylated proteins in endotoxin-stimulated RAW264.7 murine macrophages.

Nitric oxide (NO) is an omnipresent regulator of cell function in a variety of physiologic and pathophysiologic states. In part, NO exerts its actions by S-nitrosylation of target thiols, primarily in cysteine residues. Delineating the functional correlates of S-nitrosylation can begin with identification of the entire population of S-nitrososylated proteins. Recently, the biotin switch technique was developed to allow a proteomic approach to identification of the "universe" of S-nitrsoylated proteins. In this study using endotoxin-stimulated RAW264.7 murine macrophages, we have utilized the biotin-switch technique and protein sequencing to identify S-nitrosylated proteins in this setting. In contrast to other studies utilizing exogenous sources of NO, our approach utilizes endogenous NO synthesis as the basis for S-nitrosylation. Our results indicate multiple unique proteins not previously identified as S-nitrosylation targets: enolase, pyruvate kinase, elongation factor-1 and -2, plastin-2, FRAG-6, CEM-16, and SMC-6. While the ubiquitous nature of NO argues for some degrees of commonality, S-nitrosylation of unique proteins specific to endotoxin stimulated macrophages suggests regulatory mechanisms for which NO is necessary, but not sufficient.

Amidines↗
PubMed2006

A solanesyl-diphosphate synthase localizes in glycosomes of Trypanosoma cruzi.

We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass approximately 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target.

Alkyl and Aryl Transferases↗
PubMed2005

A study of the metabolites of ischemia-reperfusion injury and selected amino acids in the liver using microdialysis during transplantation.

BACKGROUND: Preservation and ischemia-reperfusion injury still impact the outcome of orthotopic liver transplantation. The authors used microdialysis with a view to monitoring its effect on graft function. METHODS: A microdialysis catheter was inserted into the graft immediately after reperfusion and perfused with an isotonic solution for 48 hr. Metabolites of the ischemia-reperfusion injury and selected amino acids were studied. There were 18 patients, with a median age of 52 years (range, 38-62 years), 8 of whom were men. Lactate, pyruvate, glycerol, and glucose levels were measured. In addition, alanine, arginine, citrulline, gamma-aminobutyric acid (GABA), glutamate, glutamine, glycine, and taurine were determined. RESULTS: All grafts functioned well. High lactate, pyruvate, and glycerol levels were observed in the immediate postoperative period. These showed a significant rapid decrease and stabilized to baseline levels. Alanine, glutamate, GABA, and taurine levels declined significantly to baseline values. Arginine levels were low immediately postreperfusion and then increased, reaching significantly higher values beyond 19 hr. CONCLUSIONS: These data may represent "normal" changes seen in the immediate posttransplant period because all grafts functioned well. Two important metabolic fates of arginine in the liver are in the detoxification of ammonia by means of the urea cycle, and in the synthesis of nitric oxide (NO). Low extracellular arginine may reflect influx of the amino acid into hepatocytes, resulting in formation of NO in the presence of inducible NO synthase or conversion to ornithine in the presence of arginase in the urea cycle. As the organ stabilizes, restriction of arginine uptake may give rise to the observed increase in extracellular arginine.

Adult↗
PubMed2006

Cloning and characterization of the dxs gene, encoding 1-deoxy-d-xylulose 5-phosphate synthase from Agrobacterium tumefaciens, and its overexpression in Agrobacterium tumefaciens.

A newly isolated gene dxs11 from Agrobacterium tumefaciens (KCCM 10413), an organism with potential for the industrial production of ubiquinone-10 (UbiQ(10)), encoding a 1-deoxy-d-xylulose 5-phosphate synthase (Dxs), was cloned in Escherichia coli and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1920bp, capable of encoding a polypeptide of 640 amino acids residues with a calculated isoelectric point of pH 5.63 and a molecular mass of 68,054Da. The homodimeric enzyme was overexpressed in E. coli and purified as an active soluble form. The enzyme required thiamine diphosphate and a divalent metal ion, either Mg(2+) or Mn(2+), for enzymatic activity. The enzyme had an optimal pH and temperature of 8.0 and 37 degrees C, respectively, with a k(cat) of 26.8s(-1) and a k(cat)/K(m) of 0.67 and 1.17s(-1)M(-1) for pyruvate and d-glyceraldehyde 3-phosphate, respectively. A. tumefaciens Dxs showed a comparable catalytic efficiency to other Dxs proteins. The dxs11 gene was transformed into A. tumefaciens KCCM 10413, and the resulting recombinant, A. tumefaciens pGX11, showed higher UbiQ(10) production (502.4mg/l) and content (8.3mg/gDCW) than A. tumefaciens KCCM 10413, by 21.9 and 23.9%, respectively. This work describes Dxs from A. tumefaciens, an organism with the potential for industrial UbiQ(10) production, and the first metabolic engineering study with the non-mevalonate pathway enzyme in A. tumefaciens.

Agrobacterium tumefaciens↗
PubMed1996

Role of nitric oxide in the circulatory failure and organ injury in a rodent model of gram-positive shock.

1. The pathological features of Gram-positive shock can be mimicked by the co-administration of two cell wall components of Staphylococcus aureus, namely lipoteichoic acid (LTA) and peptidoglycan (PepG). This is associated with the expression of the inducible isoform of nitric oxide synthase (iNOS) in various organs. We have investigated the effects of dexamethasone (which prevents the expression of iNOS protein) or aminoguanidine (an inhibitor of iNOS activity) on haemodynamics, multiple organ dysfunction syndrome (MODS) as well as iNOS activity elicited by LTA + PepG in anaesthetized rats. 2. Co-administration of LTA (3 mg kg-1, i.v.) and PepG (10 mg kg-1, i.v.) resulted in a significant increase in the plasma levels of tumour necrosis factor-alpha (TNF alpha, maximum at 90 min) as well as a biphasic fall in mean arterial blood pressure (MAP) from 120 +/- 3 mmHg (time 0) to 77 +/- 5 mmHg (at 6 h, n = 8; P < 0.05). This hypotension was associated with a significant tachycardia (4-6 h, P < 0.05) and a reduction of the pressor response elicited by noradrenaline (NA, 1 microgram kg-1, i.v., at 1-6 h; n = 8, P < 0.05). Furthermore, LTA + PepG caused time-dependent increases in the serum levels of markers of hepatocellular injury, glutamate-pyruvate-transminase (GPT) and glutamate-oxalacetate-transaminase (GOT). In addition, urea and creatinine (indicators of renal dysfunction) were increased. There was also a fall in arterial oxygen tension (PaO2), indicating respiratory dysfunction, and metabolic acidosis as shown by the significant drop in pH, PaCO2 and HCO3-. These effects caused by LTA + PepG were associated with the induction of iNOS activity in aorta, liver, kidney and lungs as well as increases in serum levels of nitrite+nitrate (total nitrite). 3. Pretreatment of rats with dexamethasone (3 mg kg-1, i.p.) at 120 min before LTA + PepG administration significantly attenuated these adverse effects as well as the increases in the plasma levels of TNF alpha caused by LTA + PepG. The protective effects of dexamethasone were associated with a prevention of the increase in iNOS activity (in aorta, liver, lung, kidney), the expression of iNOS protein (in lungs), as well as in the increase in the plasma levels of total nitrite. 4. Treatment of rats with aminoguanidine (5 mg kg-1 + 10 mg kg-1 h-1) starting at 120 min after LTA + PepG attenuated most of the adverse effects and gave a significant inhibition of iNOS activity (in various organs) as well as an inhibition of the increase in total plasma nitrite. However, aminoguanidine did not improve renal function although this agent caused a substantial inhibition of NOS activity in the kidney. 5. Thus, an enhanced formation of NO by iNOS importantly contributes to the circulatory failure, hepatocellular injury, respiratory dysfunction and the metabolic acidosis, but not the renal failure, caused by LTA + PepG in the anaesthetized rat.

Acid-Base Equilibrium↗