scholarly journals The Disruption of a Gene Encoding a Putative Arylesterase Impairs Pyruvate Dehydrogenase Complex Activity and Nitrogen Fixation in Sinorhizobium meliloti

2001 ◽  
Vol 14 (6) ◽  
pp. 811-815 ◽  
Author(s):  
María José Soto ◽  
Juan Sanjuan ◽  
José Olivares
Keyword(s):  
Nitrogen Fixation ◽  
Pyruvate Dehydrogenase ◽  
Sinorhizobium Meliloti ◽  
Pyruvate Dehydrogenase Complex ◽  
Chloroacetic Acid ◽  
Dicarboxylic Acids ◽  
Complex Activity ◽  
Gene Encoding ◽  
Physiological Importance

Nitrogen-fixing Sinorhizobium meliloti cells depend upon dicarboxylic acids as carbon and energy sources. The metabolism of these intermediate compounds of the tri-chloroacetic acid cycle is dependent upon the availability of acetyl-coenzyme A (CoA). In bacteroids, the combined activities of malic enzymes and pyruvate dehydrogenase (PDH) have been proposed to be responsible for the anaplerotic synthesis of acetyl-CoA. We obtained a S. meliloti mutant strain, PD3, in which a Tn5 insertion led to a significant decrease in the overall PDH activity. The genetic characterization of this mutant revealed that the transposon is located at the 3′ end of a gene (ada) encoding a putative arylesterase. The mutant PD3 is deficient in nitrogen fixation, which strengthens the physiological importance of PDH activity in the symbiosis of S. meliloti with alfalfa plants.

scholarly journals Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

2013 ◽  
Vol 79 (18) ◽  
pp. 5566-5575 ◽  
Author(s):  
Jens Buchholz ◽  
Andreas Schwentner ◽  
Britta Brunnenkan ◽  
Christina Gabris ◽  
Simon Grimm ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Fed Batch ◽  
Complex Activity ◽  
Gene Encoding ◽  
Content Type ◽  
Genes Encoding ◽  
Cell Densities ◽  
Dehydrogenase Complex

ABSTRACTExchange of the nativeCorynebacterium glutamicumpromoter of theaceEgene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutateddapApromoter variants led to a series ofC. glutamicumstrains with gradually reduced growth rates and PDHC activities. Upon overexpression of thel-valine biosynthetic genesilvBNCE, all strains producedl-valine. Among these strains,C. glutamicum aceEA16 (pJC4ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of thepqoandppcgenes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities,C. glutamicum aceEA16 Δpqo Δppc(pJC4ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter)l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression ofilvBNCDinstead ofilvBNCEtransformed thel-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with aYP/Sof 0.24 mol per mol of glucose and aQPof 6.9 mM per h [0.8 g/(liter × h)]. The replacement of theaceEpromoter by thedapA-A16 promoter in the twoC. glutamicuml-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate thatC. glutamicumstrains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

scholarly journals Genetic Analysis of Mutations Affecting pckA Regulation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1521-1531 ◽  
Author(s):  
Magne Østerås ◽  
Shelley A P O'Brien ◽  
Turlough M Finan
Keyword(s):  
Sinorhizobium Meliloti ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Root Nodules ◽  
Genetic Regulation ◽  
Carbon Sources ◽  
Dicarboxylic Acids ◽  
Phenotypic Analysis ◽  
Gene Encoding ◽  
Type Locus ◽  
Rapid Induction

Abstract The enzyme phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step in the gluconeogenic pathway in most organisms. We are examining the genetic regulation of the gene encoding Pck, pckA, in Rhizobium (now Sinorhizobium) meliloti. This bacterium forms N2-fixing root nodules on alfalfa, and the major energy sources supplied to the bacteria within these nodules are C4-dicarboxylic acids such as malate and succinate. R. meliloti cells growing in glucose minimal medium show very low pckA expression whereas addition of succinate to this medium results in a rapid induction of pckA transcription. We identified spontaneous mutations (rpk) that alter the regulation of pckA expression such that pckA is expressed in media containing the non-inducing carbon sources lactose and glucose. Genetic and phenotypic analysis allowed us to differentiate at least four rpk mutant classes that map to different locations on the R. meliloti chromosome. The wild-type locus corresponding to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk mutation were identified. The nucleotide sequence of this region revealed that both Tn5 insertions lay within a gene encoding a protein homologous to the Ga1R/LacI family of transcriptional regulators that are involved in metabolism.

scholarly journals Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex

1998 ◽  
Vol 329 (1) ◽  
pp. 191-196 ◽  
Author(s):  
Melissa M. BOWKER-KINLEY ◽  
I. Wilhelmina DAVIS ◽  
Pengfei WU ◽  
A. Robert HARRIS ◽  
M. Kirill POPOV
Keyword(s):  
Tissue Distribution ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
Synthetic Analogue ◽  
Complex Activity ◽  
Acetyl Coa ◽  
Tissue Specific ◽  
Specific Regulation ◽  
Dehydrogenase Complex

Tissue distribution and kinetic parameters for the four isoenzymes of pyruvate dehydrogenase kinase (PDK1, PDK2, PDK3 and PDK4) identified thus far in mammals were analysed. It appeared that expression of these isoenzymes occurs in a tissue-specific manner. The mRNA for isoenzyme PDK1 was found almost exclusively in rat heart. The mRNA for PDK3 was most abundantly expressed in rat testis. The message for PDK2 was present in all tissues tested but the level was low in spleen and lung. The mRNA for PDK4 was predominantly expressed in skeletal muscle and heart. The specific activities of the isoenzymes varied 25-fold, from 50 nmol/min per mg for PDK2 to 1250 nmol/min per mg for PDK3. Apparent Ki values of the isoenzymes for the synthetic analogue of pyruvate, dichloroacetate, varied 40-fold, from 0.2 mM for PDK2 to 8 mM for PDK3. The isoenzymes were also different with respect to their ability to respond to NADH and NADH plus acetyl-CoA. NADH alone stimulated the activities of PDK1 and PDK2 by 20 and 30% respectively. NADH plus acetyl-CoA activated these isoenzymes nearly 200 and 300%. Under comparable conditions, isoenzyme PDK3 was almost completely unresponsive to NADH, and NADH plus acetyl-CoA caused inhibition rather than activation. Isoenzyme PDK4 was activated almost 2-fold by NADH, but NADH plus acetyl-CoA did not activate above the level seen with NADH alone. These results provide the first evidence that the unique tissue distribution and kinetic characteristics of the isoenzymes of PDK are among the major factors responsible for tissue-specific regulation of the pyruvate dehydrogenase complex activity.

scholarly journals Mutation in the ntrR Gene, a Member of the vap Gene Family, Increases the Symbiotic Efficiency of Sinorhizobium meliloti

2001 ◽  
Vol 14 (7) ◽  
pp. 887-894 ◽  
Author(s):  
Boglárka Oláh ◽  
Erno Kiss ◽  
Zoltán Györgypál ◽  
Judit Borzi ◽  
Gyöngyi Cinege ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Pathogenic Bacteria ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Nifh Gene ◽  
Dry Weight ◽  
Nodule Occupancy ◽  
Gene Encoding ◽  
Symbiotic Efficiency ◽  
Host Interactions

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the “ntrPR operon,” which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

scholarly journals Sirtuin 4 Is a Lipoamidase Regulating Pyruvate Dehydrogenase Complex Activity

Cell ◽  
2014 ◽  
Vol 159 (7) ◽  
pp. 1615-1625 ◽  
Author(s):  
Rommel A. Mathias ◽  
Todd M. Greco ◽  
Adam Oberstein ◽  
Hanna G. Budayeva ◽  
Rumela Chakrabarti ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Complex Activity ◽  
Dehydrogenase Complex

scholarly journals The effects of various anions and cations on the regulation of pyruvate dehydrogenase complex activity from pig kidney cortex

1988 ◽  
Vol 253 (3) ◽  
pp. 819-825 ◽  
Author(s):  
T Pawelczyk ◽  
R A Easom ◽  
M S Olson
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Maximal Activity ◽  
Hill Coefficient ◽  
Kidney Cortex ◽  
Complex Activity ◽  
Pig Kidney ◽  
Constant Strength ◽  
Dehydrogenase Complex

The activity of pyruvate dehydrogenase complex (PDC) purified from pig kidney cortex was found to be affected by various uni- and bi-valent ions. At a constant strength of 0.13 M at pH 7.8, K+, Na+, Cl-, HCO3- and HPO4(2-) had significant effects on the activity of PDC: Na+, K+ and HPO4(2-) stimulated, but HCO3- and Cl- inhibited. The stimulatory effect of Na+ was mediated by a change in the Vmax. of PDC only, whereas K+ produced an increase in Vmax. and a change in the Hill coefficient (h). The extent of stimulation produced by HPO4(2-)4 on the activity of PDC was dependent on the concentrations of K+ and Na+. Both cations at concentrations higher than 40 mM partially prevented the effect of HPO4(2-)4. Cl- and HCO3- anions decreased the Vmax. of the enzyme and increased the S0.5 for pyruvate. The effects of Na+, K+, Cl-, HPO4(2-) and HCO3- on the activity of PDC were additive. In the presence of 80 mM-K+, 20 mM-Na+, 10 mM-HPO4(2-), 20 mM-Cl- and 20 mM-HCO3- the activity of PDC was increased by 30%, the S0.5 for pyruvate was increased from 75 to 158 microM and h was decreased from 1.3 to 1.1. Under these conditions and at 1.0 mM-pyruvate, the activity of PDC was 80% of the maximal activity achieved in the presence of these ions and 4.5 mM-pyruvate. The present study suggests that PDC may operate under non-saturating concentrations for substrate in vivo.

scholarly journals Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria

1973 ◽  
Vol 134 (2) ◽  
pp. 539-544 ◽  
Author(s):  
John M. Land ◽  
John B. Clark
Keyword(s):  
Rat Brain ◽  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Competitive Inhibitor ◽  
Brain Mitochondria ◽  
Significant Inhibition ◽  
Complex Activity ◽  
Rat Brain Mitochondria ◽  
Dehydrogenase Complex

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of 14CO2 from [1-14C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a Ki of 100μm. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.

scholarly journals Characterization of a Sinorhizobium melilotiATP-Binding Cassette Histidine Transporter Also Involved in Betaine and Proline Uptake

2000 ◽  
Vol 182 (13) ◽  
pp. 3717-3725 ◽  
Author(s):  
Eric Boncompagni ◽  
Laurence Dupont ◽  
Tam Mignot ◽  
Magne Østeräs ◽  
Annie Lambert ◽  
...  
Keyword(s):  
Glycine Betaine ◽  
Sinorhizobium Meliloti ◽  
Binding Protein ◽  
Compatible Solutes ◽  
Site Directed Mutagenesis ◽  
Uptake System ◽  
Periplasmic Binding Protein ◽  
Gene Encoding ◽  
Inhibition Of Growth

ABSTRACT The symbiotic soil bacterium Sinorhizobium melilotiuses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli(ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed inhut mutants (hutX and hutH2). Expression analysis of the hut operon determined using ahutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.

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