N]NMR determination of asparagine and glutamine nitrogen utilization for synthesis of storage protein in developing cotyledons of soybean in culture

Solid-state [¹⁵N]NMR was used to measure the use of the amide and amino nitrogens of glutamine and asparagine for synthesis of storage protein in cotyledons of soybean (Glycine max L. cv. Elf) in culture. No major discrimination in the incorporation of the amide or amino nitrogens of glutamine into protein is apparent, but the same nitrogens of asparagine are used with a degree of specificity. During the first seven days in culture with asparagine as the sole nitrogen source, the amino nitrogen donates approximately twice as much nitrogen to protein as does the amide nitrogen. The use of the amide nitrogen increases with longer periods of culture. The reduced use of the amide nitrogen was confirmed by its early appearance as ammonium in the culture medium. The amide nitrogen of asparagine was found at all times to be an essential precursor for protein because of its appearance in protein in residues whose nitrogens were not supplied by the amino nitrogen. In addition, methionine sulfoximine inhibited growth completely on asparagine, indicating that some ammonium assimilation is essential for storage protein synthesis. These results indicate that in a developing cotyledon, a transaminase reaction is of major importance in the utilization of asparagine for synthesis of storage protein and that, at least in the early stages of cotyledon development, reduced activities of ammonium-assimilating enzymes in the cotyledon tissue or in other tissues of the seed or pod may be a limiting factor in the use of asparagine-amide nitrogen.     

Inhibition of glycine decarboxylation and serine formation in tobacco by glycine hydroxamate and its effect on photorespiratory carbon flow

Glycine hydroxamate is a competitive inhibitor of glycine decarboxylation and serine formation (referred to as glycine decarboxylase activity) in particulate preparations obtained from both callus and leaf tissue of tobacco. In preparations from tobacco callus tissues, the K_i for glycine hydroxamate was 0.24 ± 0.03 millimolar and the K_m for glycine was 5.0 ± 0.5 millimolar. The inhibitor was chemically stable during assays of glycine decarboxylase activity, but reacted strongly when incubated with glyoxylate. Glycine hydroxamate blocked the conversion of glycine to serine and CO₂in vivo when callus tissue incorporated and metabolized [1-¹⁴C]glycine, [1-¹⁴C]glycolate, or [1-¹⁴C]glyoxylate. The hydroxamate had no effect on glyoxylate aminotransferase activities in vivo, and the nonenzymic reaction between glycine hydroxamate and glyoxylate did not affect the flow of carbon in the glycolate pathway in vivo. Glycine hydroxamate is the first known reversible inhibitor of the photorespiratory conversion of glycine to serine and CO₂.     

Utilization of Amino Acids as a Sole Source of Nitrogen by Obligate Chemolithoautotroph Thiobacillus ferrooxidans

An obligate chemolithoautotroph, Thiobacillus ferrooxidans API 9–3, could utilize amino acids, other than glycine, methionine and phenylalanine, as a sole source of nitrogen. However, both the growth rate and growth yield were lower than those in Fe²⁺-NH4 -salts medium, suggesting that the ammonium ion was a superior nitrogen source for the strain compared to amino acids. Methionine and phenylalanine strongly inhibited the cell growth on Fe²⁺-NH4-salts medium at 10 mm. [¹⁴C]Glycine could not be taken up into the cells, and this meant the strain could not use glycine as a sole source of nitrogen. The uptake of [¹⁴C]leucine into the cells was dependent on the presence of Fe^{2 +}. When the strain was cultured on Fe^{2 +} - leucine (lOmm)-salts medium lacking an inorganic nitrogen source for 5 days at 30°C, 83.5% and 16.5% of the cellular carbon were derived from carbon dioxide and leucine, respectively, indicating that carbon dioxide was a superior carbon source for the bacterium compared to leucine. The ammonium ion did not inhibit the utilization of leucine for cellular carbon. Leucine uptake was markedly inhibited by inhibitors of protein synthesis, such as chloramphenicol (94.3% at 1 mm), streptomycin (57.2% at 5mm) and rifampin (77.2% at 0.1 mm), respectively. Carbon dioxide uptake was also completely inhibited by chloramphenicol at 4mm. These results suggest that the transport of both amino acids and carbon dioxide into the cells was dependent on protein synthesis.     

Biosynthetically directed 2H labelling for stereospecific resonance assignments of glycine methylene groups

Stereospecific resonance assignments of the α-protons of glycine are often difficult to obtain by measurements of scalar coupling constants or nuclear Overhauser effects. Here we show that these stereospecific resonance assignments can readily be obtained by cell-free protein synthesis in D₂O, as the serine hydroxymethyltransferase, that is naturally present in E. coli cell extracts, selectively replaces the pro-2S proton of glycine by a deuterium. To encourage the conversion by serine hydroxymethyltransferase, we performed the cell-free reaction without the addition of any glycine, exploiting the capability of the enzyme to convert serine to glycine with the help of tetrahydrofolate. ¹³C-HSQC spectra of ubiquitin produced with ¹³C/¹⁵N-serine showed that about a quarter of the glycine residues derived from serine were stereospecifically deuterated. Pulse sequences are presented that select the signals from the stereospecifically deuterated glycine residues.     

The type and time of occurrence of aminopterin-induced chromosome aberrations in cultured Potorous cells

Many inhibitors of DNA synthesis have been found to induce chromosome aberrations. Our kinetic studies indicate that treatment of cellswith 10^?7M aminopterin in the presence of 10^?4M glycine, 10^?4M hypoxanthine, and 10^?4M thymidine allows continued normal cell growth. Omission of thymidine, a treatment which is known to inhibit DNA synthesis while allowing RNA and protein synthesis to continue, leads to cessation of cell growth. Treament of Potorous cell cultures with aminopterin in the presence of hypoxanthine and glycine without thymidine led to the following observations: (1) only non-exchange chromatid aberrations were formed after aminopterin treatment; (2) the aberrations were induced only in cells treated during S, and the breaks were associated with the replicating region of the chromosome; (3) breaks were observed at the first metaphase after the beginning of treatment; and (4) thymidine could reverse the chromosome-breaking action of aminopterin. A model for the molecular mechanism is suggested.     

Purine nucleotide metabolism of germinating soybean embryonic axes

Isolated soybean (Glycine max L. cv. Kent) embyronic axes metabolized [¹⁴C]glycine to ATP within the 1 hour of imbibition. Radioactivity was not detected in GTP until the 3rd hour. Throughout most of the first 24 hours of germination about 10 to 26 times as much label from [¹⁴C]glycine appears in ATP as GTP. About five times as much [¹⁴C]hypoxanthine and [¹⁴C]inosine was converted into GTP as into ATP in embryonic axes. Two independent pools of IMP appear to be used in purine nucleotide synthesis of soybean axes.     

Glycine supports in vivo reduction of nitrate in barley leaves

Glycine, a photorespiratory intermediate, enhanced the in vivo reduction of nitrate in barley (Hordeum vulgare L.) leaf slices, when included in the assay medium. Isonicotinyl hydrazide, an inhibitor of glycine oxidation, partially reduced NO₂⁻ production. The enhancement caused by glycine treatment was reversed by isonicotinyl hydrazide when both were present together in the medium. Similar effects were observed when the excised leaves were preincubated with the metabolite and the inhibitor. Glycine also partially relieved the inhibition of nitrate reduction caused by malonate, an inhibitor of the tricarboxylic acid cycle. The results support the hypothesis that glycine decarboxylation activity is a source of NADH for nitrate reductase activity.     

Glyphosate Resistance by Engineering the Flavoenzyme Glycine Oxidase

Glycine oxidase from Bacillus subtilis is a homotetrameric flavoprotein of great potential biotechnological use because it catalyzes the oxidative deamination of various amines and d-isomer of amino acids to yield the corresponding α-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid: this compound inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. In recent years, transgenic crops resistant to glyphosate were mainly generated by overproducing the plant enzyme or by introducing a 5-enolpyruvylshikimate-3-phosphate synthase insensitive to this herbicide. In this work, we propose that the enzymatic oxidation of glyphosate could be an effective alternative to this important biotechnological process. To reach this goal, we used a rational design approach (together with site saturation mutagenesis) to generate a glycine oxidase variant more active on glyphosate than on the physiological substrate glycine. The glycine oxidase containing three point mutations (G51S/A54R/H244A) reaches an up to a 210-fold increase in catalytic efficiency and a 15,000-fold increase in the specificity constant (the k_cat/K_m ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the α2-α3 loop (comprising residues 50–60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg⁵⁴ could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide.     

Structure of the Homodimeric Glycine Decarboxylase P-protein from Synechocystis sp. PCC 6803 Suggests a Mechanism for Redox Regulation

Glycine decarboxylase, or P-protein, is a pyridoxal 5′-phosphate (PLP)-dependent enzyme in one-carbon metabolism of all organisms, in the glycine and serine catabolism of vertebrates, and in the photorespiratory pathway of oxygenic phototrophs. P-protein from the cyanobacterium Synechocystis sp. PCC 6803 is an α₂ homodimer with high homology to eukaryotic P-proteins. The crystal structure of the apoenzyme shows the C terminus locked in a closed conformation by a disulfide bond between Cys⁹⁷² in the C terminus and Cys³⁵³ located in the active site. The presence of the disulfide bridge isolates the active site from solvent and hinders the binding of PLP and glycine in the active site. Variants produced by substitution of Cys⁹⁷² and Cys³⁵³ by Ser using site-directed mutagenesis have distinctly lower specific activities, supporting the crucial role of these highly conserved redox-sensitive amino acid residues for P-protein activity. Reduction of the 353–972 disulfide releases the C terminus and allows access to the active site. PLP and the substrate glycine bind in the active site of this reduced enzyme and appear to cause further conformational changes involving a flexible surface loop. The observation of the disulfide bond that acts to stabilize the closed form suggests a molecular mechanism for the redox-dependent activation of glycine decarboxylase observed earlier.     

Glycine transport in rat brain and liver mitochondria

Transport of glycine by rat brain and liver mitochondria has been investigated by both [¹⁴C]glycine uptake and swelling experiments. Glycine enters mitochondria passively down its concentration gradient by a respiratory-independent carrier-mediated process. This view is supported by the following observations: (a) glycine inside the mitochondria reaches the incubation medium concentration; (b) mitochondria swell in the presence of isoosmotic solutions of glycine in a concentration-dependent fashion; (c) the uptake of glycine is not influenced by respiratory inhibitors such as KCN or by uncouplers such as carbonylcyanide p-trifluoromethoxyphenylhydrazone; (d) initial rates of uptake approach saturation kinetics, the apparent K_m of the rat brain mitochondria for glycine being 1.7 mM and that of the liver mitochondria being 5.7 mM; (e) the rate of swelling is inhibited by methylmalonate, propionate and, at pH 6.5, by mersalyl, and (f) uptake is inhibited by phosphoserine, methylmalonate and propionate, but not by alanine or proline.     

Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells

The effects of added glycine hydroxamate on the photosynthetic incorporation of ¹⁴CO₂ into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO₂ and 20% O₂) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO₂ and 2.7% O₂). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO₂ and NH₄⁺. During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the “reverse” flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration.     

Protein and oil concentration of soybean seed cultured in vitro using nutrient solutions of differing glutamine concentration

Oil and protein are the most valuable components of soybean seed. Evidence indicates that growth and composition of soybean seed are controlled by supplies of carbon and nitrogen provided by the maternal plant to the seed, but it is difficult experimentally to control and quantify the precise amount of carbon and nitrogen provided to the seed by the whole plant. To examine whether oil and protein concentrations are affected by the supply of nitrogen to the seed, immature soybean seeds (Glycine max cv. Williams 82) were grown in vitro in nutrient solutions containing 20, 40, 60 or 80 mM of glutamine. The seeds were incubated in Erlenmeyer flasks for 8 days at 25°C. The rate of dry matter accumulation changed from 7.2 to 8.3 mg seed⁻¹ day⁻¹ as the glutamine concentration increased from 20 to 80 mM but the differences were not significant (P 0.05). Seed protein concentration increased as glutamine concentration increased from 294 mg g⁻¹ at 20 mM glutamine to as high as 445 mg g⁻¹ at 80 mM glutamine. Typical in vivo protein concentration of mature soybean seeds is about 400 mg g⁻¹. Oil and protein concentrations were negatively correlated (r²= 0.44), which indicates that oil and protein synthesis are interrelated. Protein synthesis was favoured over oil synthesis when nitrogen became more abundant. The seeds used in this study clearly demonstrated a capacity to respond to nitrogen availability with changes in seed protein concentration.     

Amino acid utilization by Gymnodinium breve

The marine dinoflagellate Gymnodinium breve utilizes erogenous amino acids for the synthesis of proteins in the light. During logarithmic growth, l-valine and l-methionine are incorporated into proteinaceous material which is retained by the cell. Glycine is also incorporated, but the glycine-containing proteins are extruded. When cells are no longer growing exponentially, all proteins that incorporated these supplied amino acids are extruded. The pronase-susceptible extruded material has a MW in excess of 300 000. When chloramphenicol is used to inhibit protein synthesis, glycine is not taken up. l-Methionine is rapidly metabolized intracellularly and is used in the synthesis of other macromolecules. l-Valine accumulates intracellularly and remains unaltered. Glycine and l-methionine appear to be transported via facilitated diffusion systems, while l-valine uptake appears to be active.     

Chemical Evidence for the Separation of Gy and AY Globin Chains by Polyacrylamide Gel Electrophoresis in Urea and Triton X-100

Polyacrylamide gel electrophoresis in urea and Triton X-100 of a hemolysate from human fetal red blood cells produces four major protein bands: α, β, and 2 γ globin chains. We have verified that the latter two are the ^Gγ and ^Aγ globin chains which have respectively glycine or alanine at position 136. After incorporation of either [³H] alanine or [³H] glycine into newly synthesized globin each y chain was isolated by preparative electrophoresis. The chains were cleaved with cyanogen bromide at methionines 55 and 133, then subjected to automated sequencing, and the residues from each sequencer turn counted. Glycine incor-poration was detected for the third turn (position 136) of the ^Gγ chain and alanine for the ^Aγ. Substantial metabolic conversion of [³H] glycine to serine and proline was also noted.     

Chemical Evidence for the Separation of Gγ and Aγ Globin Chains by Polyacrylamide Gel Electrophoresis in Urea and Triton X-100

Polyacrylamide gel electrophoresis in urea and Triton X-100 of a hemolysate from human fetal red blood cells produces four major protein bands: α, β, and 2 γ globin chains. We have verified that the latter two are the ^Gγ and ^Aγ globin chains which have respectively glycine or alanine at position 136. After incorporation of either [³H] alanine or [³H] glycine into newly synthesized globin each y chain was isolated by preparative electrophoresis. The chains were cleaved with cyanogen bromide at methionines 55 and 133, then subjected to automated sequencing, and the residues from each sequencer turn counted. Glycine incorporation was detected for the third turn (position 136) of the ^Gγ chain and alanine for the ^Aγ Substantial metabolic conversion of [³H] glycine to serine and proline was also noted.     

Aminotransfer from Alanine and Glutamate to Glycine and Serine during Photorespiration in Oat Leaves

¹⁵N-Labeled glutamate and alanine were used to examine the photorespiratory nitrogen metabolism in oat (Avena sativa L.) leaf slices. Glutamate and alanine supply amino groups for glycine formation during photorespiration. The nitrogen flux from alanine to glycine was estimated to be 3 times higher than that from glutamate. It is concluded from these results that alanine is a direct and important amino donor for photorespiratory glycine formation in oat leaves. The ¹⁵N labeling of serine was almost as high as that of glycine during the initial period of the labeling experiments. Thereafter, the ratio of ¹⁵N label in serine to ¹⁵N label in glycine declined substantially.     

Improvement of glycine biosynthesis from one-carbon compounds and ammonia catalyzed by the glycine cleavage system in vitro

Glycine cleavage system (GCS) plays a central role in one-carbon (C1) metabolism and receives increasing interest as a core part of the recently proposed reductive glycine pathway (rGlyP) for assimilation of CO₂ and formate. Despite decades of research, GCS has not yet been well understood and kinetic data are barely available. This is to a large degree because of the complexity of GCS, which is composed of four proteins (H, T, P, and L) and catalyzes reactions involving different substrates and cofactors. In vitro kinetics of reconstructed microbial multi-enzyme glycine cleavage/synthase system is desired to better implement rGlyP in microorganisms like Escherichia coli for the use of C1 resources. Here, we examined in vitro several factors that may affect the rate of glycine synthesis via the reverse GCS reaction. We found that the ratio of GCS component proteins has a direct influence on the rate of glycine synthesis, namely higher ratios of P protein and especially H protein to T and L proteins are favorable, and the carboxylation reaction catalyzed by P protein is a key step determining the glycine synthesis rate, whereas increasing the ratio of L protein to other GCS proteins does not have significant effect and the ratio of T protein to other GCS proteins should be kept low. The effect of substrate concentrations on glycine synthesis is quite complex, showing interdependence with the ratios of GCS component proteins. Furthermore, adding the reducing agent dithiothreitol to the reaction mixture not only results in great tolerance to high concentration of formaldehyde, but also increases the rate of glycine synthesis, probably due to its functions in activating P protein and taking up the role of L protein in the non-enzymatic reduction of H_ox to H_red. Moreover, the presence of some monovalent and divalent metal ions can have either positive or negative effect on the rate of glycine synthesis, depending on their type and their concentration.     

Preferential Osmolyte Accumulation: a Mechanism of Osmotic Stress Adaptation in Diazotrophic Bacteria

A common cellular mechanism of osmotic-stress adaptation is the intracellular accumulation of organic solutes (osmolytes). We investigated the mechanism of osmotic adaptation in the diazotrophic bacteria Azotobacter chroococcum, Azospirillum brasilense, and Klebsiella pneumoniae, which are adversely affected by high osmotic strength (i.e., soil salinity and/or drought). We used natural-abundance ¹³C nuclear magnetic resonance spectroscopy to identify all the osmolytes accumulating in these strains during osmotic stress generated by 0.5 M NaCl. Evidence is presented for the accumulation of trehalose and glutamate in Azotobacter chroococcum ZSM4, proline and glutamate in Azospirillum brasilense SHS6, and trehalose and proline in K. pneumoniae. Glycine betaine was accumulated in all strains grown in culture media containing yeast extract as the sole nitrogen source. Alternative nitrogen sources (e.g., NH₄Cl or casamino acids) in the culture medium did not result in measurable glycine betaine accumulation. We suggest that the mechanism of osmotic adaptation in these organisms entails the accumulation of osmolytes in hyperosmotically stressed cells resulting from either enhanced uptake from the medium (of glycine betaine, proline, and glutamate) or increased net biosynthesis (of trehalose, proline, and glutamate) or both. The preferred osmolyte in Azotobacter chroococcum ZSM4 shifted from glutamate to trehalose as a consequence of a prolonged osmotic stress. Also, the dominant osmolyte in Azospirillum brasilense SHS6 shifted from glutamate to proline accumulation as the osmotic strength of the medium increased.     

Regeneration in alfalfa tissue culture: stimulation of somatic embryo production by amino acids and N-15 NMR determination of nitrogen utilization

The production of somatic embryos in alfalfa (Medicago sativa L., cv Regen S) is increased 5- to 10-fold by alanine and proline. However, utilization of nitrogen for synthesis of protein from alanine, proline, glutamate, and glycine is not qualitatively different, even though the latter two amino acids do not increase somatic embryo formation. These determinations were made by ¹⁵N labeling with detection by nuclear magnetic resonance. Overall metabolism of the nitrogen of proline, alanine, glutamate, and glycine is also similar in two regenerating and nonregenerating genotypes with similar germplasm, except that the levels of free amino acids are consistently higher in the nonregenerating line. In addition, when regeneration is suppressed in either of the two regenerating lines, the level of intracellular free amino acids increases. This increased level of metabolites is the only direct evidence provided by analysis of nitrogen metabolism of differences between the regenerating and nonregenerating states in alfalfa.     

The interconversion of glycine and serine by plant tissue extracts

1. Extracts prepared from a variety of higher-plant tissues by ammonium sulphate fractionation were shown to catalyse the interconversion of glycine and serine. This interconversion had an absolute requirement for tetrahydrofolate and appeared to favour serine formation. 2. The biosynthesis of serine from glycine was studied in more detail with protein fractionated from 15-day-old wheat leaves. Synthesis of [¹⁴C]serine from [¹⁴C]glycine was not accompanied by labelling of glyoxylate, glycollate or formate. 3. The synthesis of serine from glycine was stimulated by additions of formaldehyde, and [¹⁴C]formaldehyde was readily incorporated into C-3 of serine in the presence of tetrahydrofolate. 4. The results are interpreted as indicating that serine biosynthesis involves a direct cleavage of glycine whereby the α-carbon is transferred via N⁵N¹⁰-methylenetetrahydrofolate to become the β-carbon of serine.