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Proline–Glutamate Chimeras in Isopeptides. Synthesis …

Proline synthesis involves a four step process starting with glutamate. One ATP and two NADPH + H+ is used per proline.

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Proline-glutamate chimeras in isopeptides. Synthesis …

Uridylyltransferase is activated by -ketoglutarateand ATP, while it is inhibited by glutamine and Pi.The following diagram summarizes the regulation of bacterial glutaminesynthetase (see text page 1035) : We can "walk through" this regulatory cascade by looking at aspecific example, namely increased levels of -ketoglutarate( reflecting a corresponding increase in NH3) levels: (1) Uridylyltransferase activity is increased (2) PII (in complex with adenylyltransferase)is uridylylated (3) Glutamine synthetase is deadenylylated (4) -ketoglutarate and NH3 form glutamine and Pi That the control of bacterial glutamine synthetase is exquisitely sensitiveto the level of the cell's nitrogen metabolites is illustrated by the fact thatthe glutamine just produced in the above cascade is now an inhibitor of furtherglutamine production. Proline, Ornithine and Arginine are derived from GlutamateThe first step involves phosphorylation of glutamate by ATP with the enzyme -glutamylkinase, followed by reduction to glutamate-5-semialdehyde which spontaneouslycyclizes (no enzyme required) to an internal Schiff base. The formation ofthe semialdehyde also requires the presence of either NADP or NADPH.The semialdehyde is a branch point, however.

Glutamic γ-semialdehyde in arginine and proline synthesis of Neurospora: ..

The marine copepod Tigriopus californicus shows rapid adjustment of intracellular proline and alanine pools in response to salinity stress. Small increases in salinity (on the order of 2 ppt or 50 mOsmoles) are sufficient to elicit proline synthesis in animals acclimated to 50% seawater. Increases in osmolarity achieved by adding organic solutes to a 50% seawater medium also elicited proline (but not alanine) synthesis. In constant 50% seawater, label from C-14-(U)-glucose is incorporated into the free alanine and glutamate (but not proline) pools; only the proline pool shows a monotonic increase in specific activity following transfer to 100% seawater. To demonstrate that glutamate is a proline precursor, C-14-(U)-glutamate was provided to animals maintained in 50% seawater (control) and animals transferred to 100% seawater; salinity change resulted in the rapid increase in alanine and proline concentrations, with proline (and not alanine) achieving specific activities similar to that of the glutamate pool. Taken together, these results indicate that small hyperosmotic stimuli result in the induction of proline synthesis from glutamate. The regulation of proline synthesis involves mechanisms discrete from those regulating the concentrations of other FAA functioning in osmotic response.

Amino Acid Metabolism, Third Edition

Salt treatment dramatically increased proline synthesis from glutamate

In S. cerevisiae, the amino acid glutamate plays an integral role in nitrogen metabolism, where it is responsible for 85% of total cellular nitrogen (; ). The ability of glutamate to serve as progenitor of 85% of total cellular nitrogen is due, in large part, to the action of glutamate dehydrogenase (GDH), which catalyzes the direct assimilation of free ammonia via reductive amination of α-ketoglutarate (). The S. cerevisiae genome contains three GDH genes encoding for enzymes that favor either the assimilation—Gdh1p & Gdh3p—or release—Gdh2p—of ammonia (see ) (; ). Although previous studies report that Gdh1p serves as the main conduit for nitrogen assimilation and glutamate biosynthesis in vivo, gdh1Δ strains are viable and do not exhibit glutamate auxotrophy suggesting that alternative pathways exist (). While the proline utilization (PUT) and glutamine synthetase-glutamate synthase (GS/GOGAT) pathways have been previously identified for their ability to facilitate glutamate biosynthesis in gdh-deficient yeast, questions remain as to the relative contribution of these pathways in mutant strains grown on various nitrogen sources (; ). A gdh-null mutant was created. The strain was able to adapt and grow on various nitrogen sources which allowed the examination of the relative contributions of the PUT and GS/GOGAT pathways to glutamate biosynthesis and nitrogen assimilation in the absence of the favored GDH pathway.

Four nitrogen sources were selected to explore the differential contributions of the PUT and GS/GOGAT pathways to glutamate biosynthesis and nitrogen assimilation in adapted gdh-deficient yeast. As a result of nitrogen catabolite repression (NCR) in S. cerevisiae, pathways for utilization of non-preferred sources of nitrogen—e.g. proline—are down-regulated in the presence of the preferred nitrogen sources ammonia, glutamine and asparagine (). In addition to NCR status, nitrogen sources in S. cerevisiae differ in their mode of utilization proceeding either through an ammonium intermediate (Ai) or via direct conversion to glutamate (Glu) (; ). Four nitrogen sources were chosen corresponding to the four possible combinations of NCR-status and pathways for utilization: (i) ammonia – Ai/repressing; (ii) glutamine – Glu/repressing; (iii) urea – Ai/de-repressing; (iv) proline – Glu/de-repressing (; ). details the relevant pathways for the utilization of these nitrogen sources including the enzymes whose activities were assayed in this study.

Department of Horticulture and Landscape Architecture

The biosynthesis of proline from glutamate involves 3 enzymes and one from BIOL 4110 at LSU

Glutamate dehydrogenase (GDH) plays a key role in the metabolism of free amino acids (FAA) in crustaceans and other metazoans. Glutamate synthesized by GDH via reductive amination is the amino group donor for alanine synthesis and the precursor required for proline synthesis. Since both proline and alanine are important intracellular osmolytes in many marine invertebrates, GDH has been widely implicated as playing a central role in response to hyperosmotic stress in these organisms. We have isolated the gene encoding a GDH homolog from the euryhaline copepod Tigriopus californicus and examined the regulation of GDH under salinity stress. The gene encodes a protein of 557 residues with 76% amino acid identity with Drosophila melanogaster GDH. The gene encodes an N-terminal mitochondrial signal sequence peptide. Only a single intron of 71 bp was found in the GDH gene in T. californicus when genomic sequences and cDNA sequences were compared. The levels of GDH mRNA do not increase during hyperosmotic stress in this copepod. The effects of salt and hyperosmotic stress on GDH enzyme activity were also investigated. GDH activities decrease with increasing NaCl concentrations in in vitro enzyme assays, while live animals exposed to hyperosmotic stress showed no change in GDH enzyme activities. Combined, these results indicate that GDH transcription and enzyme activity do not appear to function in the regulation of alanine and proline accumulation during hyperosmotic stress in T. californicus. The manner in which this important physiological process is regulated remains unknown.

N2 - Glutamate dehydrogenase (GDH) plays a key role in the metabolism of free amino acids (FAA) in crustaceans and other metazoans. Glutamate synthesized by GDH via reductive amination is the amino group donor for alanine synthesis and the precursor required for proline synthesis. Since both proline and alanine are important intracellular osmolytes in many marine invertebrates, GDH has been widely implicated as playing a central role in response to hyperosmotic stress in these organisms. We have isolated the gene encoding a GDH homolog from the euryhaline copepod Tigriopus californicus and examined the regulation of GDH under salinity stress. The gene encodes a protein of 557 residues with 76% amino acid identity with Drosophila melanogaster GDH. The gene encodes an N-terminal mitochondrial signal sequence peptide. Only a single intron of 71 bp was found in the GDH gene in T. californicus when genomic sequences and cDNA sequences were compared. The levels of GDH mRNA do not increase during hyperosmotic stress in this copepod. The effects of salt and hyperosmotic stress on GDH enzyme activity were also investigated. GDH activities decrease with increasing NaCl concentrations in in vitro enzyme assays, while live animals exposed to hyperosmotic stress showed no change in GDH enzyme activities. Combined, these results indicate that GDH transcription and enzyme activity do not appear to function in the regulation of alanine and proline accumulation during hyperosmotic stress in T. californicus. The manner in which this important physiological process is regulated remains unknown.

Highly diastereoselactive synthesis of chimeras of proline and glutamate
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L-Proline | Health Benefits and Uses of L-Proline

Using mutagenesis on a glutamate auxotroph stalled in the citric acid cycle at aconitase (in their nomenclature, glt1-1), Lundgren et al. showed that the PUT pathway could serve as a source of glutamate, as the put2-glt1-1mutant they isolated was no longer able to utilize proline as a nitrogen source (). In contrast with the results from Lundgren et al., Avendano and colleagues asserted that the GS/GOGAT pathway, in combination with the GDHs, represents the sole pathway for glutamate biosynthesis in S. cerevisiae (). In order to address this apparent discrepancy, a gdh-null mutant was created and subjected to experiments exploring the relative contribution of the PUT and GS/GOGAT pathways to glutamate biosynthesis and nitrogen assimilation following adaptation to growth on various nitrogen sources. This approach allowed for an unprecedented exploration of nitrogen assimilation in S. cerevisiae and enabled both the clarification previous findings and the discovery of previously unappreciated mechanisms for nitrogen assimilation in this organism

Amino Acid Synthesis and Metabolism

Although previous research posits a central role for GDH in glutamate metabolism and nitrogen assimilation, it does not constitute the sole pathway for these processes () (; ,). In fact, at least two other pathways have been shown to exist in S. cerevisiae for the synthesis of glutamate and assimilation of nitrogen—viz. the proline utilization (PUT) and glutamine synthetase-glutamate synthase pathways (GS/GOGAT). Proline gains entry into the cells via either the general amino acid permease, Gap1p, or the proline-specific permease, Put4p (). The enzymes that constitute the PUT pathway are proline oxidase (Put1p) and Δ1-pyrroline-5-carboxylate dehydrogenase (Put2p), which are encoded by the PUT and PUT genes, respectively (; ). In laboratory strains of S. cerevisiae, both the PUT pathway and permease genes (Gap1p and Put4p) are tightly controlled by nitrogen catabolite repression (NCR) due to the status of proline as a non-preferred nitrogen source (). The second alternative pathway for glutamate production, GS/GOGAT, consists of two enzymes acting consecutively to catalyze: (1) the ATP-dependent amidation of glutamate using free ammonia (GS) and (2) the reductive amination of α-ketoglutarate using the amide nitrogen of glutamine (GOGAT) () (; ). Ultimately, this sequence of reactions yields two molecules of glutamate for every one that enters the pathway.

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