Pyruvate carboxylase is involved in metabolism of mimosine by <Emphasis Type="Italic">Rhizobium</Emphasis> sp. strain TAL1145

The objective of this study was to determine the role of midK, which encodes a protein similar to pyruvate carboxylase, in mimosine degradation by Rhizobium sp. strain TAL1145. The midK gene is located downstream of midR in the cluster of genes for mimosine degradation in Rhizobium sp. strain TAL1145. The midK mutants of TAL1145 degraded mimosine slower than the wild-type. These mutants could utilize pyruvate as a source of carbon, indicating that there is another pyruvate carboxylase (pyc) gene in TAL1145. Two classes of clones were isolated from the library of TAL1145 by complementing a pyc mutant of Rhizobium etli, one class contained midK, while the other carried pyc. Both midK and pyc of TAL1145 complemented the midK mutant for mimosine degradation, and also the R. etli pyc mutant for pyruvate utilization. The midK-encoded pyruvate carboxylase was required for an efficient conversion of mimosine into 3-hydroxy-4-pyridone (HP). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jonathan D. Awaya and Panlada Tittabutr contributed equally to this work.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Glutamate as an inhibitor of phosphoenolpyruvate carboxylase activity in<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

The glutamate-producing bacterium, Corynebacterium glutamicum is known to possess two anaplerotic enzymes: pyruvate carboxylase (Pc) and phosphoenolpyruvate carboxylase (PEPc). In vitro, this latter enzyme appeared to be inhibited by different glutamic acid salts, whereas ammonium-glutamate had no influence on Pc activity. To investigate the in vivo relevance of PEPc activity inhibition, the intracellular concentration of glutamate was determined throughout the glutamate-producing process. The intracellular concentration was then shown to be sufficient to induce a dramatic inhibition of PEPc activity during the process. As a consequence, intracellular accumulation of glutamate could be at least partially responsible for the weak participation of PEPc within the anaplerosis activity in amino-acid-producing strains of C. glutamicum.     

Variations in ribulose 1,5-bisphosphate carboxylase protein levels,activities and subcellular distribution during photoautotrophic batch culture of Chlorogloeopsis fritschii

Ribulose 1,5-bisphosphate (RuBP) carboxylase is present in the cytoplasm and carboxysomes (polyhedral bodies) of the cyanobacterium Chlorogloeopsis fritschii. In vitro enzyme activities have been measured throughout photoautotrophic batch culture, together with RuBP carboxylase protein concentrations, determined by rocket immunoelectrophoresis. Enzyme activities and protein levels in the cytoplasmic and carboxysomal fractions varied in an apparently inverse manner during growth. The RuBP carboxylase activities per unit enzyme protein were maximal in late lag phase/early exponential phase for both cellular enzyme pools. Both rates per unit enzyme protein declined during exponential phase, cytoplasmic enzyme activity remaining consistently higher than that of the carboxysomal enzyme. Activities per unit cytoplasmic and carboxysomal enzyme protein showed very low, similar rates in late stationary phase and death phase. Dialysis experiments indicated that such changes were not due to interference in activity assays by soluble endogenous effectors. Major shifts in the subcellular distribution of RuBP carboxylase protein were found versus culture age, enzyme protein levels being predominantly carboxysomal in lag phase, mainly soluble in exponential phase and then mainly carboxysomal again in stationary/death phase. The data are discussed in terms of carboxysome function and the question of control of RuBP carboxylase synthesis in cyanobacteria.Abbreviations RuBP D-ribulose 1,5-bisphosphate - LTIB low Tris isolation buffer - HTIB high Tris isolation buffer - RIE rocket immunoelectrophoresis     

Effect of LiCl on phosphoenolpyruvate carboxylase kinase and the phosphorylation of phosphoenolpyruvate carboxylase in leaf disks and leaves of <Emphasis Type="Italic">Sorghum vulgare</Emphasis>

In the present work, the effect of LiCl on phosphoenolpyruvate carboxylase kinase (PEPCase-k), C₄ phosphoenolpyruvate carboxylase (PEPCase: EC 4.1.1.31) and its phosphorylation process has been investigated in illuminated leaf disks and leaves of the C₄ plant Sorghum vulgare. Although this salt induced severe damages to older leaves, it did not significantly alter the physiological parameters (photosynthesis, transpiration rate, intercellular CO₂ concentration) of young leaves. An immunological approach was used to demonstrate that the PEPCase-k protein accumulated rapidly in illuminated leaf tissues, consistent with the increase in its catalytic activity. In vivo, LiCl was shown to strongly enhance the light effect on PEPCase-k protein content, this process being dependent on protein synthesis. In marked contrast, the salt was found to inhibit the PEPCase-k activity in reconstituted assays and to decrease the C₄ PEPCase content and phosphorylation state in LiCl treated plants. Short-term (15 min) LiCl treatment increased IP₃ levels, PPCK gene expression, and PEPCase-k accumulation. Extending the treatment (1 h) markedly decreased IP₃ and PPCK gene expression, while PEPCase-k activity was kept high. The cytosolic protein synthesis inhibitor cycloheximide (CHX), which blocked the light-dependent up-regulation of the kinase in control plants, was found not to be active on this process in preilluminated, LiCl-treated leaves. This suggested that the salt causes the kinase turnover to be altered, presumably by decreasing degradation of the corresponding polypeptide. Taken together, these results establish PEPCase-k and PEPCase phosphorylation as lithium targets in higher plants and that this salt can provide a means to investigate further the organization and functioning of the cascade controlling the activity of both enzymes.     

In vivo photoinactivation of ribulose bisphosphate carboxylase in the gas-vacuolate cyanobacteriumMicrocystis aeruginosa

Irradiation of buoyant, gas-vacuolate cells of the cyanobacteriumMicrocystis aeruginosa by 5·10⁴ Wm^–2 of blue light for 1 h caused a 5% loss of extractable ribulose bisphosphate carboxylase activity compared to dark and red-light controls. Ribulose bisphosphate carboxylase activity was unaffected by blue light in similar experiments conducted with cells containing collapsed gas vacuoles.Abbreviations RuBP Ribulose 1,5-bis-phosphate carboxylase     

Properties of phosphoenolpyruvate carboxylase in rapidly prepared,desalted leaf extracts of the Crassulacean acid metabolism plant Mesembryanthemum crystallinum L.

Properties of phosphoenolpyruvate (PEP) carboxylase, obtained from leaves of Mesembryanthemum crystallinum L. performing Crassulacean acid metabolism (CAM), were determined at frequent time points during a 12-h light/12-h dark cycle. Leaf extracts were rapidly desalted and PEP carboxylase activity as a function of PEP concentration, malate concentration, and pH was measured within 2 min after homogenization of the tissue. Maximum velocity of PEP carboxylase was similar in the light and dark at pH 7.5 and pH 8.0. However, PEP carboxylase had as much as a 12-fold lower K _m for PEP and as much as a 20-fold higher K _i for malate during the dark than during the light periods, the magnitude of these differences being dependent on the assay pH. Assuming that enzyme properties immediately after isolation reflect the approximate state of the enzyme in vivo, these differences in enzyme properties reduce the potential for CO₂ fixation via PEP carboxylase in the light. A small decrease in cytoplasmic pH in the light would greatly magnify the above differences in day/night properties of PEP carboxylase, because the sensitivity of PEP carboxylase to inhibition by malate increased with decreasing pH. Properties of PEP carboxylase were also studied in plants exposed to short-term perturbations of the normal 12-h light/12-h dark cycle (e.g., prolonged light period, prolonged dark period). Under all light/dark regimes, there was a close correlation between change in properties of PEP carboxylase and changes of the tissue from acidification to deacidification, and vice versa. Changes in properties of PEP carboxylase were not merely light/dark phenomena because they were also observed in plants exposed to continuous light or dark. the data indicate that, during CAM, PEP carboxylase exists in two stages which differ in their capacity for net malate synthesis. The physiologically-active state is distinguished by a low K _m for PEP and a high K _i for malate and favors malate synthesis. The physiologically-inactive state has a high K _m for PEP and a low K _i for malate and exists during periods of deacidification and other periods lacking synthesis of malic acid.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPC PEP carboxylase - RuBP ribulose 1,5-bisphosphate - RH relative humidity     

Immunocytochemical study of phosphoenolpyruvate carboxylase in nodulated Alnus glutinosa

The activities of phosphoenolpyruvate carboxylase (PEP carboxylase, EC 4.1.1.3.1) have been investigated in various organs of young nodulated Alnus glutinosa. The root nodules exhibited the highest specific enzyme activity when compared with the one in roots and leaves. Furthermore, in the root nodules the PEP carboxylase was predominantly localized in the cytosol of the large cortical cells containing the endophyte vesicles.Abbreviations PEP carboxylase phosphoenolpyruvate carboxylase - MDH malate dehydrogenase - PVP polyvinylpyrrolidone - PBS phosphate buffer saline     

Phosphoenolpyruvate carboxylase in soybean root nodules: An immunochemical study

The activity of phosphoenolpyruvate carboxylase (E.C. 4.1.1.31) strongly increased during the maturation of soybean (Glycine max L. Weber) root-nodules. By using a specific immune serum it was shown that this increase was the consequence of an elevated population of enzyme molecules whose appearance preceded the emergence of nitrogen fixing capacity. Whether or not the phenomenon could be ascribed to the formation of a specific isoenzyme is not known. The location of the enzyme was also investigated. Immunocyto-fluorescence experiments established that phosphoenolpyruvate carboxylase was present in the cytoplasmic compartment of both infected and uninfected cells of nodules.Abbreviation PEPCase phosphoenolpyruvate carboxylase     

Hydrogen-stimulated CO2 fixation and coordinate induction of hydrogenase and ribulosebiphosphate carboxylase in a H2-uptake positive strain of Rhizobium japonicum

H₂-uptake positive strains (122 DES and SR) and H₂-uptake negative strains SR2 and SR3 of Rhizobium japonicum were examined for ribulosebisphosphate (RuBP) carboxylase and H₂-uptake activities during growth conditions which induced formation of the hydrogenase system. The rate of ¹⁴CO₂ uptake by hydrogenase-derepressed cells was about 6-times greater in the presence than in the absence of H₂. RuBP carboxylase activity was observed in free-living R. japonicum strains 122 DES or SR only when the cells were derepressed for their hydrogenase system. Hydrogenase and RuBP carboxylase activities were coordinately induced by H₂ and both were repressed by added succinate. Hydrogenase-negative mutant strains SR2 and SR3 derived from R. japonicum SR showed no detecyable RuBP carboxylase activities under hydrogenase derepression conditions. No detectable RuBP carboxylase was observed in bacteroids formed by H₂-uptake positive strains R. japonicum 122 DES or SR. Propionyl CoA carboxylase activity was consistently observed in extracts of cells from free-living cultures of R. japonicum but activity was not appreciably influenced by the addition of H₂. Neither phosphoenolpyruvate carboxylase nor phosphoenolpyruvate carboxykinase activity was detected in extracts of R. japonicum.Abbreviations RuBP Ribulose 1,5-bisphosphate - (Na₂EDTA) (Ethylenedinitrilo)-tetraacetic acid, disodium salt - (propionyl CoA) Propionyl coenzyme A - (PEP) Phosphoenolpyruvate - (GSH) Reduced glutathione - (Tricine) N-tris(hydroxymethyl)-methylglycine     

Hormonal Regulation of Photosynthetic Enzymes in Cotton under Water Stress

Activities of ribulose bisphosphate carboxylase/oxygenase (RuBPCO), phosphoenolpyruvate carboxylase (PEPC), and carbonic anhydrase (CA) were determined in leaves of cotton (Gossypium hirsutum L. cv. H-777) subjected to 8-d waterlogging (WL) at the vegetative stage, or to drought (D) at the reproductive stage, or to interaction of both stresses. The soil moisture of control plants was kept at field capacity. One day prior to stress various growth hormones (5 μM) were sprayed up to runoff. WL reduced RuBPCO and CA activities, while PEPC activity increased. Upon D, RuBPCO and PEPC activities were reduced while CA activity was increased. Imposition of both stresses increased activities of all three enzymes. Effect of stresses on enzyme activity was alleviated by benzylaminopurine (BAP), but indol-3-yl-acetic acid was more promoting under interactive stress. No CA activity with BAP was observed during interactive stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

Overexpression of Acetyl-CoA Carboxylase Gene of <Emphasis Type="Italic">Mucor rouxii</Emphasis> Enhanced Fatty Acid Content in <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Malonyl-CoA is an essential precursor for fatty acid biosynthesis that is generated from the carboxylation of acetyl-CoA. In this work, a gene coding for acetyl-CoA carboxylase (ACC) was isolated from an oleaginous fungus, Mucor rouxii. According to the amino acid sequence homology and the conserved structural organization of the biotin carboxylase, biotin carboxyl carrier protein, and carboxyl transferase domains, the cloned gene was characterized as a multi-domain ACC1 protein. Interestingly, a 40% increase in the total fatty acid content of the non-oleaginous yeast Hansenula polymorpha was achieved by overexpressing the M. rouxii ACC1. This result demonstrated a significant improvement in the production of fatty acids through genetic modification in this yeast strain.     

Activity and quantity of ribulose bisphosphate carboxylase-and phosphoenolpyruvate carboxylase-protein in two Crassulacean acid metabolism plants in relation to leaf age,nitrogen nutrition,and point in time during a day/night cycle

Activity of ribulose 1,5-bisphosphate (RuBP) carboxylase in leaf extracts of the constitutive Crassulacean acid metabolism (CAM) plant Kalanchoe pinnata (Lam.) Pers. decreased with increasing leaf age, whereas the activity of phosphoenolpyruvate (PEP) carboxylase increased. Changes in enzyme activities were associated with changes in the amount of enzyme proteins as determined by immunochemical analysis, sucrose density gradient centrifugation, and SDS gel electrophoresis of leaf extracts. Young developing leaves of plants which received high amounts of NO ₃ ^- during growth contained about 30% of the total soluble protein in the form of RuBP carboxylase; this value declined to about 17% in mature leaves. The level of PEP carboxylase in young leaves of plants at high NO ₃ ^- was an estimated 1% of the total soluble protein and increased to approximately 10% in mature leaves, which showed maximum capacity for dark CO₂ fixation. The growth of plants at low levels of NO ₃ ^- decreased the content of soluble protein per unit leaf area as well as the extractable activity and the percentage contribution of both RUBP carboxylase and PEP carboxylase to total soluble leaf protein. There was no definite change in the ratio of RuBP carboxylase to PEP carboxylase activity with a varying supply of NO ₃ ^- during growth. It has been suggested (e.g., Planta 144, 143–151, 1978) that a rhythmic pattern of synthesis and degradation of PEP carboxylase protein is involved in the regulation of -carboxylation during a day/night cycle in CAM. No such changes in the quantity of PEP carboxylase protein were observed in the leaves of Kalanchoe pinnata (Lam.) Pers. or in the leaves of the inducible CAM plant Mesembryanthemum crystallinum L.Abbreviations CAM Crassulacean acid metabolism - RuBP ribulose 1,5-bisphosphate - PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate     

The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression

Summary The ppc gene of Corynebacterium glutamicum encoding phosphoenolpyruvate (PEP) carboxylase was isolated by complementation of a ppc mutant of Escherichia coli using a cosmid gene bank of chromosomal c. glutamicum DNA. By subsequent subcloning into the plasmid pUC8 and deletion analysis, the ppc gene could be located on a 3.3 kb SalI fragment. This fragment was able to complement the E. coli ppc mutant and conferred PEP carboxylase activity to the mutant. The complete nucleotide sequence of the ppc gene including 5 and 3 flanking regions has been determined and the primary structure of PEP carboxylase was deduced. The sequence predicts a 919 residue protein product (molecular weight of 103154) which shows 34% similarity with the respective E. coli enzyme. Present address: Institut für Biotechnologie 1 der Kernforschungsanlage, Postfach 1913, D-5170 Jülich, Federal Republic of Germany     

Cloning of the gene for phosphoenolpyruvate carboxylase from Azotobacter chroococcum, an enzyme important in aerobic nitrogen fixation

Summary Azotobacter chroococcum Fos 189 is a Tn1-induced mutant which, unlike the parent strain MCD1, does not fix nitrogen in air when provided with glucose or pyruvate as sole carbon sources. Fos 189 showed 5% of parental activity for phosphoenolpyruvate carboxylase though PEP synthetase activity was normal. The A. chroococcum phosphoenolpyruvate carboxylase (ppc) gene was isolated after complementation of an appropriate Escherichia coli mutant using a broad host range gene bank prepared from A. chroococcum genomic DNA. The gene was localised by transposon mutagenesis and subcloning on a minimum DNA fragment of 6.6 kb. Broad host range plasmids containing the A. chroococcum ppc gene complemented the mutation in Fos 189 thereby restoring aerotolerant nitrogen fixation.     

Differences in the Activation State of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase in Barley,Pea, and Wheat at Two Altitudes

Activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) is an important parameter determining the rate of net photosynthesis (P _N) in situ for which no information is available with reference to altitude. We analyzed activation state along with P _N in three plant species and their cultivars grown at low (LA, 1 300 m) and high (HA, 4 200 m) altitudes. No significant change in P _N and the initial activity of RuBPCO was obtained with reference to altitude. However, activation state of RuBPCO was reduced significantly in the HA plants as compared to the LA ones. Hence low partial pressure of CO₂ prevailing at HA might be responsible for the lower activation state of RuBPCO.     

An efficient succinic acid production process in a metabolically engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> strain

A Corynebacterium glutamicum strain (ΔldhA-pCRA717) that overexpresses the pyc gene encoding pyruvate carboxylase while simultaneously exhibiting a disrupted ldhA gene encoding l-lactate dehydrogenase was investigated in detail for succinic acid production. Succinic acid was shown to be efficiently produced at high-cell density under oxygen deprivation with intermittent addition of sodium bicarbonate and glucose. Succinic acid concentration reached 1.24 M (146 g l⁻¹) within 46 h. The yields of succinic acid and acetic acid from glucose were 1.40 mol mol⁻¹ (0.92 g g⁻¹) and 0.29 mol mol⁻¹ (0.10 g g⁻¹), respectively. The succinic acid production rate and yield depended on medium bicarbonate concentration rather than glucose concentration. Consumption of bicarbonate accompanied with succinic acid production implied that added bicarbonate was used for succinic acid synthesis.     

On the cellular localization of phosphoenolpyruvate carboxylase in Sorghum leaves

The localization of phosphoenol pyruvate carboxylase (EC 4.1.1.3.1.) in the leaf cells of Sorghum vulgare was investigated by using three techniques: the conventional aqueous and non aqueous methods gave conflicting results; the immunocytochemical techniques clearly showed that the enzyme is predominantly located in the cytoplasm of mesophyll cells.Abbreviations PEP phosphoenol pyruvate - PAG polyacrylamide gel - NADP MDH NADP malate dehydrogenase - FITC fluorescein isothiocyanate - SAB serum albumine bovine - DTT dithiothreitol - MDH malate dehydrogenase - ME malic enzyme - PBS phosphate buffer saline - PAP peroxidase anti-peroxidase     

Changes in the activities of carbon metabolizing enzymes with pod development in lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> L.)

Activities of some key enzymes of carbon metabolism sucrose synthase, acid and alkaline invertase, phosphoenol pyruvate carboxylase, malic enzyme and isocitrate dehydrogenase were investigated in relation to the carbohydrate status in lentil pods. Sucrose remained the dominant soluble sugar in the pod wall and seed, with hexoses (glucose and fructose) present at significantly lower levels. Sucrose synthase is the predominant sucrolytic enzyme in the developing seeds of lentil (Lens culinaris L.). Acid invertase was associated with pod elongation and showed little activity in seeds. Sucrose breakdown was dominated by alkaline invertase during the development of podwall, while both the sucrose synthase and alkaline invertase were active in the branch of inflorescence. A substantial increase of sucrolytic enzymes was observed at the time of maximum seed filling stage (10–20 DAF) in lentil seed. The pattern of activity of sucrose synthase highly paralleled the phase of rapid seed filling and therefore, can be correlated with seed sink strength. It seems likely that the fruiting structures of lentil utilize phosphoenol pyruvate carboxylase for recapturing respired carbon dioxide. Higher activities of isocitrate dehydrogenase and malic enzyme in the seed at the time of rapid seed filling could be effectively linked to the deposition of protein reserves.     

Effects of rapidly imposed water deficit on photosynthetic parameters of three C<Subscript>4</Subscript> grasses

Water deficit, when rapidly imposed on three C₄ grasses of the different metabolic subtypes, Paspalum dilatatum Poiret (NADP-malic enzyme), Cynodon dactylon (L.) Pers (NAD-malic enzyme) and Zoysia japonica Steudel (phosphoenolpyruvate carboxykinase), caused decreases in photosynthetic rates, in the quantum yield of PS II and photochemical quenching, and in the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC). The results provide evidence for non-stomatal limitations of photosynthesis differing in nature between the three species.