The purification and kinetic properties of biophosphoglycerate synthase from horse red blood cells.

Bisphosphoglycerate synthase from horse red cells has been purified to apparent homogeneity by a simple and efficient new procedure incorporating chromatography on a column of Sepharose 4B derivatized with blue dextran. The enzyme is similar to the human red cell synthase in subunit size. It is phosphorylated by either glycerate-1,3-P₂ or glycerate-2,3-P₂ to form a phosphoenzyme with the acid-lability of a histidyl phosphate. In addition to the synthase activity (glycerate-1,3-P₂ → glycerate-2,3-P₂), k_cat 12.5 s^?1, the enzyme has bisphosphoglycerate phosphatase activity in the presence of glycolate-2-P (glycerate-2,3-P₂ → glycerate-P + P_i), k_cat 2.6 s^?1 and phosphoglycerate mutase activity (3-PGA ? 2-PGA), k_cat 1.7 s^?1. The energy of activation for the synthase reaction is 9.38 kcal/mol. Lineweaver-Burk plots of the kinetic data are parallel lines. In contrast intersecting patterns were obtained from similar experiments done with the human red cell enzyme. Further investigation is required to explain these differences. This enzyme may function as both synthase and phosphatase for bisphosphoglycerate in the red blood cell.     

Characterization of Poly-γ-Glutamate Hydrolase Encoded by a Bacteriophage Genome: Possible Role in Phage Infection of Bacillus subtilis Encapsulated with Poly-γ-Glutamate

Some Bacillus subtilis strains, including natto (fermented soybeans) starter strains, produce a capsular polypeptide of glutamate with a γ-linkage, called poly-γ-glutamate (γ-PGA). We identified and purified a monomeric 25-kDa degradation enzyme for γ-PGA (designated γ-PGA hydrolase, PghP) from bacteriophage ΦNIT1 in B. subtilis host cells. The monomeric PghP internally hydrolyzed γ-PGA to oligopeptides, which were then specifically converted to tri-, tetra-, and penta-γ-glutamates. Monoiodoacetate and EDTA both inhibited the PghP activity, but Zn²⁺ or Mn²⁺ ions fully restored the enzyme activity inhibited by the chelator, suggesting that a cysteine residue(s) and these metal ions participate in the catalytic mechanism of the enzyme. The corresponding pghP gene was cloned and sequenced from the phage genome. The deduced PghP sequence (208 amino acids) with a calculated M_r of 22,939 was not significantly similar to any known enzyme. Thus, PghP is a novel γ-glutamyl hydrolase. Whereas phage ΦNIT1 proliferated in B. subtilis cells encapsulated with γ-PGA, phage BS5 lacking PghP did not survive well on such cells. Moreover, all nine phages that contaminated natto during fermentation produced PghP, supporting the notion that PghP is important in the infection of natto starters that produce γ-PGA. Analogous to polysaccharide capsules, γ-PGA appears to serve as a physical barrier to phage absorption. Phages break down the γ-PGA barrier via PghP so that phage progenies can easily establish infection in encapsulated cells.     

In vitro evaluation of new functional properties of poly-γ-glutamic acid produced by Bacillus subtilis D7

We investigated the functionality of poly-γ-glutamic acid (γ-PGA), which is produced by Bacillus subtilis D7, for its potential applications in medicine and cosmetics. The γ-PGA had angiotensin-converting enzyme (ACE) inhibition activity. ACE inhibition activity was dependent on the γ-PGA concentration; the highest ACE inhibition activity was observed at 1.25 mg/l of γ-PGA. IC₅₀ (0.108 mg/ml) of the γ-PGA was lower than that of standard ACE inhibitory drug, N-[(S)-mercapto-2-methylpropionyl]-L-proline (0.247 mg/ml). The γ-PGA also had water-holding capacity and hygroscopicity. Furthermore, the γ-PGA inhibited growth of some pathogenic bacteria, including Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumonia and Esherichia coli. The γ-PGA exhibited a good metal adsorption capacity; Cr (VI) adsorption capacity of γ-PGA increased with decreasing pH, and the maximal adsorption was observed at pH 2. Our results suggest that γ-PGA may be expected to be widely applied in cosmetics, biomedical and environmental industries with the feature of being less harmful to humans and the environment.     

Glycerate-3-phosphate, produced by CO2 fixation in the Calvin cycle, is critical for the synthesis of the D1 protein of photosystem II

We demonstrated recently that, in intact cells of Chlamydomonas reinhardtii, interruption of CO₂ fixation via the Calvin cycle inhibits the synthesis of proteins in photosystem II (PSII), in particular, synthesis of the D1 protein, during the repair of PSII after photodamage. In the present study, we investigated the mechanism responsible for this phenomenon using intact chloroplasts isolated from spinach leaves. When CO₂ fixation was inhibited by exogenous glycolaldehyde, which inhibits the phosphoribulokinase that synthesizes ribulose-1,5-bisphosphate, the synthesis de novo of the D1 protein was inhibited. However, when glycerate-3-phosphate (3-PGA), which is a product of CO₂ fixation in the Calvin cycle, was supplied exogenously, the inhibitory effect of glycolaldehyde was abolished. A reduced supply of CO₂ also suppressed the synthesis of the D1 protein, and this inhibitory effect was also abolished by exogenous 3-PGA. These findings suggest that the supply of 3-PGA, generated by CO₂ fixation, is important for the synthesis of the D1 Protein. It is likely that 3-PGA accepts electrons from NADPH and decreases the level of reactive oxygen species, which inhibit the synthesis of proteins, such as the D1 protein.     

Purification and Characterization of Phosphoenolpyruvate Carboxylase from Developing Seeds of Brassica

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified to apparent homogeneity with about 29% recovery from developing seeds of Brassica using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sepharose CL-6S. The purified enzyme with mol wt of about 400 kD exhibited maximum activity at pH 8.0. The enzyme had an absolute requirement for a divalent cation which was satisfied by Mg²⁺. The enzyme showed typical hyperbolic kinetics with PEP and HCO^?3 with Km of 0.125 and 0.104 mM, respectively. Glu-6-P could activate the enzyme, whereas other phosphate esters such as fru-1, 6-P₂, L-glycerophosphate and 3-PGA did not have any effect on the enzyme activity. Noneof the amino acids at 5 mM concentration had any significant effect on the enzyme activity. Nucleotide monophosphates and diphosphates did not inhibit the enzyme significantly, whereas ATP inhibited the enzyme activity. Oxaloacetate and malate inhibited the enzyme non-competitively with respect to PEP with Ki values of 0.127 and 1.25 mM, respectively. The enzyme activity in vivo seems to be regulated ’Tlainly by availability of its substrate and activation by glu-6-P, both of which are supplied through glycolysis.     

Glutamic acid independent production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of pgsBCA genes

A new glutamic acid independent poly-γ-glutamic acid (γ-PGA) producing strain, which was identified as Bacillusamyloliquefaciens LL3 by analysis of 16S rDNA and gyrase subunit A gene (gyrA), was isolated from fermented food. The product had a molecular weight of 470, 801 and l-glutamate monomer content of 98.47%. The pre-optimal medium, based on single-factor tests and orthogonal design, contained 50 g/L sucrose, 2 g/L (NH₄)₂SO₄, 0.6 g/L MgSO₄, and provided well-balanced changes in processing parameters and a γ-PGA yield of 4.36 g/L in 200 L system. The γ-PGA synthetase genes pgsBCA were cloned from LL3, and successfully expressed by pTrcLpgs vector in Escherichia coli JM109, resulting the synthesis of γ-PGA without glutamate. This study demonstrates the designedly improved yield of γ-PGA in 200 L system and the first report of pgsBCA from glutamic acid independent strain, which will benefit the metabolized mechanism investigation and the wide-ranging application of γ-PGA.     

Regulation of ADPglucose pyrophosphorylase in cucumber plants infected with the cucumber mosaic virus

ADPglucose pyrophosphorylase level and mechanisms regulating its activity were studied in cucumber plants infected with the cucumber mosaic virus at the stage of chronic infection. Studies carried out with partially purified preparations of the enzyme have shown that there was no substantial difference in the regulatory influence of the ratio 3-PGA/P₁, or in the number of binding sites of the effectors on the enzyme, but that the virus infection reduced the level of the enzyme in the tissues to 74% of the control and the 3-PGA/P₁ ratio to one half which resulted in a further decrease in ADPglucose pyrophosphorylase activity. In crude homogenate prepared from diseased plants, activity of the enzyme was reduced to 42% of the healthy control. The level of UDPglucose pyrophosphorylase was three times higher in cucumber leaf tissues than the level of ADPglucose pyrophosphorylase which was inhibited by both 3-PGA and P₁. Inhibitory effects of both these effectors were cumulated. The enzyme isolated from healthy plants was inhibited by inorganic phosphate more strongly than the enzyme isolated from diseased plants. UDPglucose pyrophosphorylase activity was increased in crude homogenate of diseased plants to 127% of the healthy control when the level of the enzyme was the same in the tissues of both healthy and diseased plants which was presumably connected with the enhanced rate of sucrose catabolism.     

Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34⁺ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G₀/G₁ cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G₀/G₁ cell cycle arrest with activation of p53. Infection of cord blood CD34⁺ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.     

Optimization of γ-polyglutamic acid synthesis using response surface methodology of a newly isolated glutamate dependent <Emphasis Type="Italic">Bacillus velezensis</Emphasis> Z3

A new glutamate-dependent γ-polyglutamic acid (γ-PGA) producer Z3 isolated from soil samples in Daxinganling forest region of China was identified, and its optimal medium components were investigated using response surface methodology. Strain Z3 was identified as Bacillus velezensis by physiology and biochemistry and 16S rDNA sequence analysis. This is the first report of glutamate-dependent B. velezensis with the ability to synthesize γ-PGA. Then, the optimum γ-PGA yield (5.58 g/L) was achieved with glutamate 86 g/L, glucose 36 g/L, yeast extract powder 5.5 g/L, and NaH₂PO₄ 7.5 g/L. Furthermore, activities of enzymes participating in glutamate synthesis were assessed, and the results showed that lower ketoglutaric dehydrogenase activity (KGDH) and higher glutamate dehydrogenase activity (GDH) resulted in higher γ-PGA yield. Identification of glutamate-dependent γ-PGA producer named B. velezensis Z3 enriches microbiological resources with γ-PGA-producing capacity. B. velezensis optimization of nutrients and analysis of enzymes activities will not only help to increase γ-PGA productivity but also to understand the γ-PGA synthesis mechanism in B. velezensis Z3.     

Hydrogenase-Mediated Activities in Isolated Chloroplasts of Chlamydomonas reinhardii

Isolated intact chloroplasts of Chlamydomonas reinhardii were found to catalyze photoreduction of CO₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when adapted under an atmosphere of H₂ demonstrating the association of a hydrogenase and anaerobic adaptation system with these plastids. The specific activity of photoreduction was approximately one third that detected in cells and protoplasts. Photoreduction was found to have a lower osmoticum optimum relative to aerobically maintained chloroplasts (50 millimolar versus 120 millimolar mannitol). 3-Phosphoglycerate (3-PGA) stimulated photoreduction up to a peak at 0.25 millimolar beyond which inhibition was observed. In the absence of 3-PGA, inorganic phosphate had no effect on photoreduction but in the presence of 3-PGA, inorganic phosphate also stimulated the reaction. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone inhibited photoreduction but inhibition by the former could be partially overcome by exogenously added ATP. The intact plastid can also catalyze photoevolution of H₂ while lysed chloroplast extracts catalyzed the reduction of methyl viologen by H₂. Both reactions occurred at rates approximately one-third of those found in cells. The oxyhydrogen reaction in the presence or absence of CO₂ was not detected.     

Characterization of an autonomously activated plant ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch biosynthesis in plants and changes in its catalytic and/or allosteric properties can lead to increased starch production. Recently, a maize (Zea mays)/potato (Solanum tuberosum) small subunit mosaic, MP [Mos(1–198)], containing the first 198 amino acids of the small subunit of the maize endosperm enzyme and the last 277 amino acids from the potato tuber enzyme, was expressed with the maize endosperm large subunit and was reported to have favorable kinetic and allosteric properties. Here, we show that this mosaic, in the absence of activator, performs like a wild-type AGPase that is partially activated with 3-phosphoglyceric acid (3-PGA). In the presence of 3-PGA, enzyme properties of Mos(1–198)/SH2 are quite similar to those of the wild-type maize enzyme. In the absence of 3-PGA, however, the mosaic enzyme exhibits greater activity, higher affinity for the substrates, and partial inactivation by inorganic phosphate. The Mos(1–198)/SH2 enzyme is also more stable to heat inactivation. The different properties of this protein were mapped using various mosaics containing smaller portions of the potato small subunit. Enhanced heat stability of Mos(1–198) was shown to originate from five potato-derived amino acids between 322 and 377. These amino acids were shown previously to be important in small subunit/large subunit interactions. These five potato-derived amino acids plus other potato-derived amino acids distributed throughout the carboxyl-terminal portion of the protein are required for the enhanced catalytic and allosteric properties exhibited by Mos(1–198)/SH2.     

Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities

An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-¹⁴C,5-³H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate V_c/V_o ratios from the expression A/(B-A) where A and B represent the ³H/¹⁴C isotope ratios of doubly labeled RuBP and 3-PGA, and V_c and V_o represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-¹⁴C]glucose and [6-³H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.     

RuBP carboxylase determination by enzymic estimation of D-3-PGA formed

RuBPcarboxylase activity was measured in extracts of barley (Hordeum Vulgare L., cv. HOP) seedlings both with the standard radiometric method and by measuring D-3-phosphoglyceric acid formed enzymically in a two stage assay. In the different conditions used, characterized by different NaHCO₃ concentrations, different pH and the presence and absence of oxygen, essentially the same ratio of D-3-PGA formed per ¹⁴CO₂ fixed was obtained. This ratio respected the known stoichiometry of two molecules of D-3-PGA formed per CO₂ fixed. It is suggested that measurement of D-3-PGA enzymically in a two stage assay can be routinely used for the determination of RuBP case activity instead of the radiometric method. The advantages and the validity of the method are discussed.     

Investigation on enzymatic degradation of γ-polyglutamic acid from Bacillus subtilis NX-2

The preparation of γ-polyglutamic acid (γ-PGA) from Bacillus subtilis NX-2 has been previously investigated, and its depolymerization during the batch culture was studied in this paper. The results suggested that the γ-PGA depolymerase was present and active extracellularly in the culture. The ywtD gene from B. subtilis NX-2, encoding the γ-PGA depolymerase was cloned and expressed in Escherichia coli. The YwtD protein was purified by metal-chelating affinity chromatography. YwtD was proved to be an endo-hydrolase enzyme and exhibited a remarkable activity in γ-PGA degradation at a wide range of temperature (30–40 °C) and pH (5.0–8.0). On an optimal condition of 30 °C and pH 5.0, an efficient γ-PGA enzymatic degradation was achieved. The molecular weight of γ-PGA could be reduced within the range of 1000–20 kDa and the polydispersity also decreased as a function of depolymerization time. Therefore, a controllable degradation of γ-PGA could be available by enzymatic depolymerization.     

Phytochrome B-dependent regulation of reductive phase of photosynthetic carbon assimilation

The experiments were conducted with 10-day-old seedlings of wheat (Triticum aestivum L). Phytochrome B was activated using an array of light diodes emitting light in the red spectral region (RL) and inactivated by an array of light diodes emitting far-red light (FRL). At the end of the night dark period (8 h), activity of the chloroplastic GAP-dehydrogenase complex (the sequence of reactions: 3-PGA → 1,3-PGA → 3-GAP) was 1.0?1.2 μmol of oxidized NADPH/(min g fr wt of the leaf). When the leaves of intact plants were exposed to a maximal dose of RL (20 min at 17.5 kJ/m²), enzyme activity rose by 100–120%. Longer exposure to RL (30 and 40 min) did not cause further activation. Successive exposure to RL and FRL (20 min at 3.0 kJ/m²) completely negated a stimulatory effect of RL. It was shown that as little as 5-min-long exposure to RL increased the rate of 3-GAP formation by 20–25%, and enzyme activity rose linearly when radiation dose was elevated. Determination of the lifetime of RL-activated state by its decrease in plants placed in darkness showed that decay occurred with τ_1/2 of 50?60 min when RL was switched off. Thus, a phytochrome B-induced regulation of reducing enzyme complex governing the reductive pentose phosphate cycle was discovered. Judging from the kinetics of attenuation of the activated state, phytochrome B apparently does not affect de novo synthesis of the enzyme. Since the investigated metabolic process consists of two coupled reactions controlled by kinase and dehydrogenase, the place and mechanism of action of the phytochrome system remain unknown.     

Production and optimization of poly-γ-glutamic acid by Bacillus subtilis BL53 isolated from the Amazonian environment

The aims of this research were to screen and characterize a new microbial source of γ-PGA, to optimize aspects of culture conditions and medium composition using central composite design and response surface methodologies. The influence of bioreactor stirring rates on the production of γ-PGA was also investigated and the oxygen volumetric mass transfer coefficients (k _La) were established. The most productive strain was identified by 16S rDNA analysis as Bacillus subtilis, and its γ-PGA production in rotatory shaker was threefold increased under optimized conditions (37 °C, pH 6.9, and 1.22 mM Zn²⁺), compared to conventional medium. In bioreactor, the γ-PGA production was further increased, reaching 17 g l^?1, 70 % higher than shaker cultures. γ-PGA production showed high dependency on oxygen transfer. At k _La of 210 h^?1, the cultivation time could be reduced to 48 h, about 50 % of the time required for operations at k _La 55 h^?1.     

Physical and Kinetic Properties and Regulation of the NAD Malic Enzyme Purified from Leaves of Crassula argentea

The NAD malic enzyme has been purified to near homogeneity from the leaves of Crassula argentea Thunb. The enzyme has two subunits, one of 59,000 daltons, and one of 62,000 daltons. In native gels stained for activity, the enzyme appears to exist in the dimeric, tetrameric, and predominantly the octameric forms.

The enzyme uses either Mg²⁺ or Mn²⁺ as the required divalent cation, and utilizes NADP at a rate less than 20% of that with NAD. With Mn²⁺ the K_m for malate²⁻ is lower than with Mg²⁺, but V_max is lower than with Mg²⁺. In the forward (malate-decarboxylating) direction with NAD, the kinetic parameters are essentially like those observed for the enzyme from C₃ plants. In the reverse reaction, run with Mn²⁺, the activity is 1.5% of that in the forward reaction. The equilibrium constant is 1.1 × 10⁻³ molar.

The kinetic mechanism of the reaction, at least in the forward direction, is sequential, with apparently random binding of all reaction components. Product inhibition patterns confirm this.

The enzyme displays a strong hysteretic lag, which is shortened by high enzyme concentrations, high substrate concentrations, and the presence of the product NADH.

The enzyme is activated by coenzyme A with K_a = 4 micromolar. AMP also shows competitive activation, with K_a = 24 micromolar. The activation by coenzyme A and AMP is additive, implying separate sites for their binding. Phosphoenolpyruvate activates the reaction at low (micromolar) concentrations, but higher concentrations of phosphoenolpyruvate cause deactivation. Fumarate²⁻ is a strong activator, with K_a = 0.3 millimolar. Fructose-1,6-bisphosphate activates the enzyme, but its most pronounced effect is in shortening the lag. Citrate is a competitive inhibitor of malate, with K_i = 4.9 millimolar.

     

Pyruvate orthophosphate dikinase of c(3) seeds and leaves as compared to the enzyme from maize

Pyruvate orthophosphate dikinase (PPDK) was found in various immature seeds of C₃ plants (wheat, pea, green bean, plum, and castor bean), in some C₃ leaves (tobacco, spinach, sunflower, and wheat), and in C₄ (maize) kernels. The enzyme in the C₃ plants cross-reacts with rabbit antiserum against maize PPDK. Based on protein blot analysis, the apparent subunit size of PPDK from wheat seeds and leaves and from sunflower leaves is about 94 kdaltons, the same as that of the enzyme from maize, but is slightly less (about 90 kdaltons) for the enzyme from spinach and tobacco leaves. The amount of this enzyme per mg of soluble protein in C₃ seeds and leaves is much less than in C₄ leaves. PPDK is present in kernels of the C₄ plant, Zea mays in amounts comparable to those in C₄ leaves.

Regulatory properties of the enzyme from C₃ tissues (wheat) are similar to those of the enzyme from C₄ leaves with respect to in vivo light activation and dark inactivation (in leaves) and in vivo cold lability (seeds and leaves).

Following incorporation of ¹⁴CO₂ by illuminated wheat pericarp and adjoining tissue for a few seconds, the labeled metabolites were predominantly products resulting from carboxylation of phosphoenolpyruvate, with lesser labeling of compounds formed by carboxylation of ribulose 1,5-bisphosphate and operation of the reductive pentose phosphate cycle of photosynthesis. PPDK may be involved in mechanisms of amino acid interconversions during seed development.