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1.
Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum 总被引:1,自引:0,他引:1
Sabine Merkelbach Johanna Gehlen Martin Denecke Heinz-Josef Hirsch Fritz Kreuzaler 《Plant molecular biology》1993,23(4):881-888
A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC-
Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes. 相似文献
2.
The crystal structure of an archaeal‐type phosphoenolpyruvate carboxylase from Clostridium perfringens has been determined based on X‐ray data extending to 3 Å. The asymmetric unit of the structure includes two tetramers (each a dimer‐of‐dimers) of the enzyme. The precipitant, malonate, employed for the crystallization is itself a weak inhibitor of phosphoenolpyruvate carboxylase and a malonate molecule is seen in the active‐site in the crystal structure. The allosteric binding sites for aspartate (an inhibitor) and glucose‐6‐phosphate (an activator) observed in the Escherichia coli and Zea mays phosphoenolpyruvate carboxylase structures, respectively, are not conserved in the C. perfringens structure. Aspartate inhibits the C. perfringens enzyme competitively with respect to the substrate, Mg++. phosphoenolpyruvate. A mechanism for inhibition is proposed based on the structure and sequence comparisons with other archaeal‐type phosphoenolpyruvate carboxylases with differing sensitivity to inhibition by aspartate. Proteins 2011; © 2011 Wiley‐Liss, Inc. 相似文献
3.
Soohyun Park Kwang Suk Chang EonSeon Jin Seung Pil Pack Jinwon Lee 《Bioprocess and biosystems engineering》2013,36(1):127-131
A new phosphoenolpyruvate carboxylase (PEPC) gene of Dunaliella salina is identified using homology analysis was conducted using PEPC gene of Chlamydomonas reinhardtii and Arabidopsis thaliana. Recombinant E. coli SGJS115 with increased production of malate and oxaloacetate was developed by introducing codon-optimized phosphoenolpyruvate carboxylase2 (OPDSPEPC2) gene of Dunaliella salina. E. coli SGJS115 yielded a 9.9 % increase in malate production. In addition, E. coli SGJS115 exhibited two times increase in the yield of oxaloacetate over the E. coli SGJS114 having identified PEPC2 gene obtained from Dunaliella salina. 相似文献
4.
The in-situ inter- and intracellular localization patterns of phosphoenolpyruvate (PEP) and ribulose 1,5-bisphosphate (RuBP) carboxylases in green leaves of severalPanicum species were investigated using an indirect immunofluorescence technique. Four species were examined and compared:P. miliaceum (C4),P. bisulcatum (C3), andP. decipiens andP. milioides (C3–C4 intermediates which have Kranz-like leaf anatomy and reduced photorespiration). In the C4
Panicum, PEP carboxylase was located in the cytosol of the mesophyll cells and RuBP carboxylase was restricted to the bundle-sheath chloroplasts. In contrast, in the C3
Panicum species, PEP carboxylase was found throughout the leaf chlorenchyma, in both the cytosol and chloroplasts, and RuBP carboxylase was located in the chloroplasts. For the C3–C4 intermediate plants, the patterns depended on the species examined. ForP. decipiens, the in-situ localization of both carboxylases was similar to that described forP. bisulcatum and other C3 plants. However, inP. milioides, PEP carboxylase was found exclusively in the cytosol of the mesophyll cells, as inP. miliaceum and other C4 species, whereas RuBP carboxylase was distributed in both the mesophyll and bundle-sheath chloroplasts.Abbreviations PEP
phosphoenolpyruvate
- RuBP
ribulose 1,5-bisphosphate 相似文献
5.
Effect of Overexpression of Actinobacillus succinogenes Phosphoenolpyruvate Carboxykinase on Succinate Production in Escherichia coli
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Pil Kim Maris Laivenieks Claire Vieille J. Gregory Zeikus 《Applied microbiology》2004,70(2):1238-1241
Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc::kan, PEPCK overexpression increased succinate production 6.5-fold. 相似文献
6.
Khidir W. Hilu 《Plant Systematics and Evolution》1994,189(3-4):247-257
Echinochloa (Poaceae) includes two domesticated species,Echinochloa utilis (Japanese barnyard millet) andE. frumentacea (Indian sawa millet) and 20–30 wild species. The two millets are morphologically very variable and overlap in spikelet and inflorescence characteristics. Both species are hexaploids based on x = 9. Cytogenetic studies point to the hexaploid wild speciesE. crusgalli andE. colona as possible progenitors ofE. utilis andE. frumentacea, respectively. The tetraploidE. oryzoides is considered as a possible genome donor to wild and domesticated barnyard millet. Markers from Random Amplified Polymorphic DNA method were used to assess the proposed phylogeny and examine the genetic diversity in both domesticated and wild species. The data were analyzed numerically.Echinochloa utilis andE. frumentacea appear very distinct, but grouped withE. crusgalli andE. colona, respectively. The tetraploidE. oryzoides show strong genetic affinity to theE. utilis—E. crusgalli group. The data are in general agreement with the cytogenetic information; however, some disagreements on the interpretation of some of the cytogenetic information is raised. The variability in DNA markers observed in the domesticated species, particularlyE. frumentacea, points to the feasibility of using RAPD markers in cultivar fingerprinting and breeding programs of these millets. 相似文献
7.
Sorghum vulgare phosphoenolpyruvate carboxylase (PEPC) and Lactococcus lactis pyruvate carboxylase (PYC) were overexpressed in Escherichia coli concurrently to improve the production of succinate, a valuable industrial specialty chemical. This coexpression system was also applied to E. coli mutant strains strategically designed by inactivating the competing pathways of succinate formation. The highest level of succinate production was observed in E. coli strains coexpressing both PEPC and PYC when compared with E. coli strains individually overexpressing either PEPC or PYC. Lactate production was also significantly reduced with PEPC and PYC coexpression. Lactate and acetate pathways were inactivated to eliminate the competing pathways of succinate formation. Results showed that inactivation of both the lactate and acetate pathways with the coexpression of PEPC and PYC was most effective in improving succinate production. Inactivating the lactate or acetate pathway alone only caused a majority of the carbon flux to shift to other metabolites rather than succinate. Coexpression of PEPC and PYC was also applied to an E. coli mutant strain deficient in lactate dehydrogenase and pyruvate:formate lyase that accumulated a substantial amount of the intermediate metabolite pyruvate during growth. Results showed that PEPC and PYC coexpression was effective in depleting pyruvate accumulation and increasing the production of metabolites. 相似文献
8.
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 相似文献
9.
10.
Likun Cheng Jing Wang Xiubao Zhao Huanhuan Yin Haitian Fang Chuwen Lin Shasha Zhang Zhiqiang Shen Chunguang Zhao 《Biotechnology progress》2020,36(6):e3058
Phage infection is common during the production of L-threonine by E. coli, and low L-threonine production and glucose conversion percentage are bottlenecks for the efficient commercial production of L-threonine. In this study, 20 antiphage mutants producing high concentration of L-threonine were obtained by atmospheric and room temperature plasma (ARTP) mutagenesis, and an antiphage E. coli variant was characterized that exhibited the highest production of L-threonine Escherichia coli ([E. coli] TRFC-AP). The elimination of fhuA expression in E. coli TRFC-AP was responsible for phage resistance. The biomass and cell growth of E. coli TRFC-AP showed no significant differences from those of the parent strain (E. coli TRFC), and the production of L-threonine (159.3 g L−1) and glucose conversion percentage (51.4%) were increased by 10.9% and 9.1%, respectively, compared with those of E. coli TRFC. During threonine production (culture time of 20 h), E. coli TRFC-AP exhibited higher activities of key enzymes for glucose utilization (hexokinase, glucose phosphate dehydrogenase, phosphofructokinase, phosphoenolpyruvate carboxylase, and PYK) and threonine synthesis (glutamate synthase, aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase) compared to those of E. coli TRFC. The analysis of metabolic flux distribution indicated that the flux of threonine with E. coli TRFC-AP reached 69.8%, an increase of 16.0% compared with that of E. coli TRFC. Overall, higher L-threonine production and glucose conversion percentage were obtained with E. coli TRFC-AP due to increased activities of key enzymes and improved carbon flux for threonine synthesis. 相似文献
11.
Leaf inner structure and immunogold localization of some key enzymes involved in carbon metabolism in CAM plants 总被引:1,自引:1,他引:0
There are relatively few reports on the leaf structure and in
situ immunolocalization of carbon metabolism enzymes in
crassulacean acid metabolism (CAM) plants, compared with reports on C4
plants. The leaf inner structure and the subcellular location of some key
CAM enzymes for a phosphoenolpyruvate carboxykinase (PCK) CAM species,
Ananas comosus, and three malic enzyme (ME) CAM
species, Mesembryanthemum crystallinum, Kalanchoe
daigremontiana, and K. pinnata, was
investigated by immunogold labelling and electron microscopy in this study.
The leaves of these species had few intercellular air spaces in the
mesophyll. A large vacuole occupied the mesophyll cells, and many vesicles
of various sizes occurred in the cytosol. Immunocytochemical study revealed
that labelling was present for phosphoenolpyruvate carboxylase in the
cytosol and for ribulose-1,5-bisphosphate carboxylase/oxygenase in the
chloroplasts of the mesophyll cells in all species. No specific labelling
for pyruvate orthophosphate dikinase (PPDK) was observed in the PCK-CAM
species. In the ME-CAM species, the patterns of labelling for PPDK
differed. In M. crystallinum labelling for PPDK was
present only in the chloroplasts, whereas in the two
Kalanchoe species it occurred in the cytosol
as well as in the chloroplasts. These results suggest that the subcellular
localization of PPDK varies with ME-CAM species, in contrast to the
conventional belief that it is localized in the chloroplasts.Key
words: Crassulacean acid metabolism, immunolocalization, leaf
inner structure, phosphoenolpyruvate carboxylase, pyruvate orthophosphate
dikinase.
相似文献
12.
Immunological studies on phosphoenolpyruvate carboxylase in Sedum species with crassulacean acid metabolism or C3 photosynthesis 总被引:1,自引:1,他引:0
Abstract From Sedum morganianum, which is a plant species known to have constitutive crassulacean acid metabolism (CAM), phosphoenolpyruvate (PEP) carboxylase (E.C.4.1.1.31) has been extracted and purified by (NH4)2SC4 precipitation, ion exchange chromatography and gel electrophoresis. A specific antibody to this purified enzyme was obtained by immunization of a rabbit. This antibody was used to compare the antigen–antibody reaction of PEP-carboxylases prepared from other Sedum species including constitutive, facultative and non-CAM plants. The experiments revealed partial immunological indentity of PEP-carboxylases obtained from the different sources. 相似文献
13.
Bernhard J. Eikmanns Max T. Follettie Martin U. Griot Anthony J. Sinskey 《Molecular & general genetics : MGG》1989,218(2):330-339
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 相似文献
14.
The occurrence of coliforms in a small water course was shown to increase by a factor of thirty six below the outfall of a
sewage treatment plant. Speciation of the bacteria from above and below the sewer outfall showed thatEscherichia coli andEnterobacter species predominated. Drug resistance levels were significant in microorganisms from both sampling sites and the occurrence
of a significant number of multiple-resistant microorganisms, particularlyE. coli, is reported. BothE. coli andEnterobacter species from below the sewer outfall show a statistically significant increase in resistance to ampicillin andE. coli from below the outfall also shows a statistically significant increase in resistance to sulphamethoxazole as compared with
isolates from above the outfall. 相似文献
15.
Summary The phosphoenolpyruvate carboxylase gene (ppc) of Escherichia coli K-12 was cloned on the multi-copy plasmid pLG339. Plasmid pST101, which carried a 4.3-kb SalI fragment, was introduced into Serratia marcescens T-1165, which carried the seven regulatory mutations for three aspartokinases and two homoserine dehydrogenases. Strain T-1165[pST101] produced phosphoenolpyruvate carboxylase at a rate 26 times higher than the control strain T-1165[pLG339]. While T-1165[pST101] produced 63 mg/ml l-threonine in a medium containing sucrose and urea, whereas T-1165 only produced 52 mg/ml. 相似文献
16.
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. 相似文献
17.
Barbara L. Randolph-Anderson Nicholas W. Gillham John E. Boynton 《Journal of molecular evolution》1989,29(1):68-88
Summary Antibodies to individual chloroplast ribosomal (r-)proteins ofChlamydomonas reinhardtii synthesized in either the chloroplast or the cytoplasm were used to examine the relatedness ofChlamydomonas r-proteins to r-proteins from the spinach (Spinacia oleracea) chloroplast,Escherichia coli, and the cyanobacteriumAnabaena 7120. In addition,35S-labeled chloroplast r-proteins from large and small subunits ofC. reinhardtii were coelectrophoresed on 2-D gels with unlabeled r-proteins from similar subunits of spinach chloroplasts,E. coli, andAnabaena to compare their size and net charge. Comigrating protein pairs were not always immunologically related, whereas immunologically related r-protein pairs often did not comigrate but differed only slightly in charge and molecular weight. In constrast, when35S-labeled chloroplast r-proteins from large and small subunits of a closely related speciesC. smithii were coelectrophoresed with unlabeledC. reinhardtii chloroplast r-proteins, only one pair of proteins from each subunit showed a net displacement in mobility.Analysis of immunoblots of one-dimensional SDS and two-dimensional urea/SDS gels of large and small subunit r-proteins from these species revealed more antigenic conservation among the four species of large subunit r-proteins than small subunit r-proteins.Anabaena r-proteins showed the greatest immunological similarity toC. reinhardtii chloroplast r-proteins. In general, antisera made against chloroplast-synthesized r-proteins inC. reinhardtii showed much higher levels of cross-reactivity with r-proteins fromAnabaena, spinach, andE. coli than did antisera to cytoplasmically synthesized r-proteins. All spinach r-proteins that cross-reacted with antisera to chloroplast-synthesized r-proteins ofC. reinhardtii are known to be made in the chloroplast (Dorne et al. 1984b). FourE. coli r-proteins encoded by the S10 operon (L2, S3, L16, and L23) were found to be conserved immunologically among the four species. Two of the large subunit r-proteins, L2 and L16, are essential for peptidyltransferase activity. The third (L23) and two otherE. coli large subunit r-proteins (L5 and L27) that have immunological equivalents among the four species are functionally related to but not essential for peptidyltransferase activity. 相似文献
18.
The occurrence of both C3 and C4 photosynthetic characteristics in a single Zea mays plant 总被引:1,自引:0,他引:1
The activities of the carboxylating enzymes ribulose-1,5-biphosphate (RuBP) carboxylase and phosphoenolpyruvate (PEP) carboxylase in leaves of three-week old Zea mays plants grown under phytotron conditions were found to vary according to leaf position. In the lower leaves the activity of PEP carboxylase was lower than that of RuBP carboxylase, while the upper leaves exhibited high levels of PEP carboxylase. Carbon dioxide compensation points and net photosynthetic rates also differed in the lower and upper leaves. Differences in the fine structure of the lowermost and uppermost leaves are shown. The existence of both the C3 and C4 photosynthetic pathways in the same plant, in this and other species, is discussed.Abbreviations PEP
phosphoenolpyruvate
- RuBP
ribulose-1,5-biphosphate 相似文献
19.
A detailed comparison of green leaf phosphoenolpyruvate carboxylases from the C(4)-species Atriplex spongiosa and the C(3)-species Atriplex hastata revealed significant physical and kinetic differences. The two alloenzymes can be separated by anion exchange chromatography but have comparable molecular weights (350,000). Maximal velocity estimates were 38.0 and 1.48 micromoles per minute per milligram of chlorophyll for the carboxylases of A. spongiosa and A. hastata, respectively. Km phosphoenolpyruvate estimates were 0.49 and 0.08 mm for the C(4)A. spongiosa and C(3)A. hastata and the Km Mg estimates were 0.33 mm for the C(4) species and 0.017 mm for the C(3) species. The activity of the phosphoenolpyruvate carboxylase of A. spongiosa is more sensitive to chloride and phosphate than the phosphoenolpyruvate carboxylase of A. hastata, but both are equally sensitive to Mg chelating substances such as ATP, ADP, and citrate if assayed at their respective Km Mg values. A survey of the phosphoenolpyruvate carboxylases from 18 C(4) and C(3) species resulted in mean maximal velocity estimates of 29.0 +/- 13.2 and 1.50 +/- 0.57 micromoles per minute per milligram of chlorophyll for the C(4) species and C(3) species, respectively. Km phosphoenolpyruvate estimates were 0.59 +/- 0.35 mm and 0.14 +/- 0.07 mm for the C(4) and C(3), and Km Mg estimates were 0.50 +/- 0.30 and 0.097 +/- 0.057 mm for C(4) and C(3). All differences between means were significant at the 0.01 confidence level, supporting our hypothesis that the phosphoenolpyruvate carboxylase alloenzymes of C(4) and C(3) plants are functionally different and are associated with different photosynthetic roles. Both function in the photosynthetic production of C(4) acids, the phosphoenolpyruvate carboxylase of C(4) species largely producing malate or aspartate (or both) as a photosynthetic intermediate and the phosphoenolpyruvate carboxylase of C(3) species producing malate or aspartate (or both) as a photosynthetic product. 相似文献
20.
Soohyun Park Soohye Hong Seung Pil Pack Jinwon Lee 《Bioprocess and biosystems engineering》2014,37(2):331-335
Phosphoenolpyruvate carboxylase (PEPC) of Photobacterium profundum SS9 can be expressed and purified using the Escherichia coli expression system. In this study, a codon-optimized PEPC gene (OPPP) was used to increase expression levels. We confirmed OPPP expression and purified it from extracts of recombinant E. coli SGJS117 harboring the OPPP gene. The purified OPPP showed a specific activity value of 80.3 U/mg protein. The OPPP was stable under low temperature (5–30 °C) and weakly basic conditions (pH 8.5–10). The enzymatic ability of OPPP was investigated for in vitro production of oxaloacetate using phosphoenolpyruvate (PEP) and bicarbonate. Only samples containing the OPPP, PEP, and bicarbonate resulted in oxaloacetate production. OPPP production system using E. coli could be a platform technology to produce high yields of heterogeneous gene and provide the PEPC enzyme, which has high enzyme activity. 相似文献