Purification, properties and assay of D-ribulose 5-phosphate 3-epimerase from human erythrocytes.
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ABSTRACT:
SUBMITTER: Karmali A
PROVIDER: S-EPMC1154406 | biostudies-other | 1983 Jun
REPOSITORIES: biostudies-other
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Project description:Chloroplast ribulose-5-phosphate-3-epimerase (RPE) is a critical enzyme involved in the Calvin-Benson cycle and oxidative pentose phosphate pathways in higher plants. Three Arabidopsis rpe mutants with reduced level of RPE were identified through their high NPQ (nonphotochemical quenching) phenotype upon illumination, and no significant difference of plant size was found between these rpe mutants and WT (wild type) plants under growth chamber conditions. A decrease in RPE expression to a certain extent leads to a decrease in CO2 fixation, V cmax and J max. Photosynthetic linear electron transport was partially inhibited and activity of ATP synthase was also decreased in the rpe mutants, but the levels of thylakoid protein complexes and other Calvin-Benson cycle enzymes in rpe mutants were not affected. These results demonstrate that some degree of reduction in RPE expression decreases carbon fixation in chloroplasts, which in turn feedback inhibits photosynthetic electron transport and ATP synthase activity due to the photosynthetic control. Taken together, this work provides evidence that RPE plays an important role in the Calvin-Benson cycle and influences the photosynthetic capacity of chloroplasts.
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Project description:Pentose phosphate pathway (PPP) plays a crucial role in the maintenance of NADPH/NADP+ homeostasis and provides protection against oxidative stress through detoxification of the reactive oxygen species. Ribulose-5-phosphate epimerase (RPE) participates in catalysis of the interconversion of ribulose-5-phosphate (Ru5P) to xylulose-5-phosphate (Xu5P) during PPP, however the structural attributes of this enzyme are still underexplored in many human pathogens including leishmanial parasites. The present study focuses upon cloning, purification and characterization of RPE of Leishmania donovani (LdRPE) using various biophysical and structural approaches. Sequence analysis has shown the presence of trypanosomatid-specific insertions at the N-terminus that are absent in humans and other eukaryotes. Gel filtration chromatography indicated recombinant LdRPE to exist as a dimer in the solution. Circular dichroism studies revealed a higher alpha helical content at physiological pH and temperature that comparatively varies with changing these parameters. Additionally, intrinsic fluorescence and quenching studies of LdRPE have depicted that tryptophan residues are mainly buried in the hydrophobic regions, and the recombinant enzyme is moderately tolerant to urea. Moreover, homology modeling was employed to generate the three-dimensional structure of LdRPE followed by molecular docking with the substrate, product, and substrate analogues. The modeled structure of LdRPE unravelled the presence of conserved active site residues as well as a single binding pocket for the substrate and product, while an in silico study suggested binding of substrate analogues into a similar pocket with more affinity than the substrate. Additionally, molecular dynamics simulation analysis has deciphered complexes of LdRPE with most of the ligands exhibiting more stability than its apo form and lesser fluctuations in active site residues in the presence of ligands. Altogether, our study presents structural insights into leishmanial RPE that could provide the basis for its implication to develop potent antileishmanials.
| S-EPMC8756792 | biostudies-literature
Project description:The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these structural findings.
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Project description:Ribulose 5-phosphate (Ru5P) and guanosine 5'-triphosphate (GTP) are two key precursors of riboflavin, whereby Ru5P is also a precursor of GTP. Ribulose 5-phosphate 3-epimerase (Rpe) catalyzes the conversion of ribulose 5-phosphate into xylulose 5-phosphate. Inactivation of Rpe can reduce the consumption of Ru5P, enhancing the carbon flux toward riboflavin biosynthesis. Here we investigated the effect of mutation of rpe and other related genes on riboflavin production, physiological and metabolic phenotypes in Bacillus subtilis LY (BSLY). Introducing single nucleotide deletion (generated BSR) or nonsense mutation (generated BSRN) on the genomic copy of rpe, resulting in more than fivefold increase of riboflavin production over the parental strain. BSR process 62% Rpe activity, while BSRN lost the entire Rpe activity and had a growth defect compared with the parent strain. BSR and BSRN exhibited increases of the inosine and guanine titers, in addition, BSRN exhibited an increase of inosine 5'-monophosphate titer in fermentation. The transcription levels of most oxidative pentose phosphate pathway and purine synthesis genes were unchanged in BSR, except for the levels of zwf and ndk, which were higher than in BSLY. The production of riboflavin was increased to 479.90 ± 33.21 mg/L when ribA was overexpressed in BSR. The overexpression of zwf, gntZ, prs, and purF also enhanced the riboflavin production. Finally, overexpression of the rib operon by the pMX45 plasmid and mutant gnd by pHP03 plasmid in BSR led to a 3.05-fold increase of the riboflavin production (977.29 ± 63.44 mg/L), showing the potential for further engineering of this strain.
| S-EPMC8359813 | biostudies-literature
Project description:H(2)O(2) is commonly generated in biological habitats by environmental chemistry and by cellular immune responses. H(2)O(2) penetrates cells, disrupts metabolism, and blocks growth; it therefore is of interest to identify the major cellular molecules that H(2)O(2) damages and the strategies by which cells protect themselves from it. We used a strain of Escherichia coli that lacks catalases and peroxidases to impose protracted low-grade H(2)O(2) stress. Physiological analysis indicated that the pentose-phosphate pathway, in particular, was poisoned by submicromolar intracellular H(2)O(2). Assays determined that ribulose-5-phosphate 3-epimerase (Rpe) was specifically inactivated. In vitro studies demonstrated that Rpe employs a ferrous iron atom as a solvent-exposed cofactor and that H(2)O(2) rapidly oxidizes this metal in a Fenton reaction. The oxidized iron is released immediately, causing a loss of activity. Most Rpe proteins could be reactivated by remetallation; however, a small fraction of proteins were irreversibly damaged by each oxidation cycle, and so repeated cycles of oxidation and remetallation progressively led to permanent inactivation of the entire Rpe pool. Manganese import and iron sequestration are key elements of the H(2)O(2) stress response, and we found that manganese can activate Rpe in vitro in place of iron, converting the enzyme to a form that is unaffected by H(2)O(2). Indeed, the provision of manganese to H(2)O(2)-stressed cells protected Rpe and enabled the pentose-phosphate pathway to retain function. These data indicate that mononuclear iron enzymes can be primary targets of H(2)O(2) stress and that cells adapt by shifting from iron- to manganese-centered metabolism.
| S-EPMC3069151 | biostudies-literature
Project description:1. Diaminopimelate epimerase from a soluble extract of Bacillus megaterium N.C.I.B. 7581 was purified about 25-fold by fractionation with ammonium sulphate and chromatography on calcium phosphate gel-cellulose. The product was impure but was unstable on further purification. 2. Quantitative assay methods for the enzyme were devised in which meso- or ll-diaminopimelic acid may be the substrate. 3. Between 25 degrees and 45 degrees at pH7.0 enzyme action leads to an equilibrium mixture containing 65% meso-isomer and 35% ll-isomer. 4. The initial rate of epimerization was 2-3 times as fast with ll-diaminopimelic acid as substrate as with the meso-isomer; a number of other amino acids were not racemized by the enzyme. The Michaelis constants at 37 degrees were 6.7mm (ll-isomer) and 100mm (meso-isomer); with both substrates enzyme activity was maximal at pH7-8. The relative rates of epimerization of ll-diaminopimelic acid at 25 degrees , 37 degrees and 45 degrees were 0.77:1.00:1.15. 5. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was needed as an activator of the purified enzyme. 6. Carbonylbinding reagents and several other compounds did not inhibit diaminopimelate epimerase. 7. Pyridoxal phosphate did not stimulate enzymic activity even in preparations that had been almost completely freed of derivatives of vitamin B(6) (as shown by microbiological assay).
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Project description:d-Allulose has potential as a low-calorie sweetener which can suppress fat accumulation. Several enzymes capable of d-allulose production have been isolated, including d-tagatose 3-epimerases. Here, we report the isolation of a novel protein from Methylomonas sp. expected to be a putative enzyme based on sequence similarity to ketose 3-epimerase. The synthesized gene encoding the deduced ketose 3-epimerase was expressed as a recombinant enzyme in Escherichia coli, and it exhibited the highest enzymatic activity toward l-ribulose, followed by d-ribulose and d-allulose. The X-ray structure analysis of l-ribulose 3-epimerase from Methylomonas sp. (MetLRE) revealed a homodimeric enzyme, the first reported structure of dimeric l-ribulose 3-epimerase. The monomeric structure of MetLRE is similar to that of homotetrameric l-ribulose 3-epimerases, but the short C-terminal α-helix of MetLRE is unique and different from those of known l-ribulose 3 epimerases. The length of the C-terminal α-helix was thought to be involved in tetramerization and increasing stability; however, the addition of residues to MetLRE at the C terminus did not lead to tetramer formation. MetLRE is the first dimeric l-ribulose 3-epimerase identified to exhibit high relative activity toward d-allulose.
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Project description:1. GAMMA-Glutamylcyclotransferase was purified 10000-fold from human erythrocytes. 2. The purification steps involved fractionation with (NH4)(2)SO(4) and chromatography on Sephadex G-75, DEAE-cellulose and hydroxyapatite. The purified enzyme was found to be homogeneous on density-gradient polyacrylamide-gel electrophoresis. 3. The maximum reaction rate was observed at pH9.0 and the apparent Km value for gamma-glutamyl-L-alanine was 2.2mM. 4. The molecular weight (25250) of the purified enzyme agreed well with the value (25500) in fresh haemolysates, indicating no apparent structural modification of the enzyme during purification. However, rapid processing of the blood through the initial (NH4)(2)SO(4) and Sephadex-chromatography steps was required to prevent formation of a high-molecular-weight aggregate with substantially lower specific activity. 5. gamma-Glutamylcyclotransferase catalyses the formation of 5-oxoproline from gamma-glutamyl dipeptides. The role of this enzyme in erythrocytes is of particular interest, because gamma-glutamyl-L-cysteine serves as a substrate for both gamma-glutamylcyclotransferase and glutathione synthetase. Thus the cyclotransferase could modulate glutathione synthesis.
| S-EPMC1185795 | biostudies-other
Project description:A new procedure for the isolation of homogeneous human 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) is described in which the enzyme is purified 35000-fold and in 65-74% yield. The specific activity of the purified enzyme, 24 units/mg, is the highest yet reported. An efficient stage for the removal of haemoglobin is incorporated in the method, which has general application to the purification of other erythrocyte enzymes. The erythrocyte dehydratase (Mr 285 000) is made up of eight apparently identical subunits of Mr 35 000. The enzyme is sensitive to oxygen, and its activity is maintained by the presence of thiols such as dithioerythritol. Zn2+ is obligatory for enzyme activity, the apoenzyme being essentially inactive (approximately equal to 12% of control) when assayed in buffers devoid of Zn2+. Addition of Zn2+ to the apoenzyme restores activity as long as the sensitive thiol groups are fully reduced; optimal stimulation occurs between 100 and 300 microM-Zn2+. The human enzyme is inhibited by Pb2+ in a non-competitive fashion [KiI (dissociation constant for E X S X Pb2+ complex) = 25.3 +/- 3.0 microM; KiS (dissociation constant for E X Pb2+ complex) = 9.0 +/- 2.0 microM]. Modification of thiol groups, inactivation by oxidation, alkylation or reaction with thiophilic reagents demonstrates the importance of sensitive thiol groups for full enzymic activity.
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Project description:Arginase was isolated from human liver and erythrocytes. The purification procedure used acetone precipitation, heat-treatment, (NH4)2SO4 precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-200 in the presence of 2-mercaptoethanol. Both enzymes migrated to the anode at pH8.3 on polyacrylamide-gel electrophoresis. After incubation at pH8.0 and 37 degrees C the purified anionic liver arginase migrated to the cathode on polyacrylamide-gel electrophoresis. It is assumed that the multiple forms of the enzyme reported in the literature are partly artifacts of the purification procedure. The liver arginase showed a mol.wt. of 107000 determined by gel filtration and a sedimentation coefficient of 5.9S. Treatment of the liver enzyme with 0.25% sodium dodecyl sulphate at pH10 demonstrated an oligomeric structure of the enzyme with a mol.wt. of the subunit of 35000. The kinetic properties determined for the purified liver arginase showed an optimum pH of 9.3 and an optimal MnCl2 concentration of 2mM. The Km for L-arginine was 10.5 mM and for L-canavanine 50mM, and L-lysine exhibited a competitive type of inhibition with a Ki of 4.4mM. L-Homoarginine was not a substrate for liver arginase.
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