Proline biosynthesis in a proline-accumulating barley mutant

A barley (Hordeum vulgare L.) mutant, R5201, selected for resistance to 4? mM trans-4-hydroxyproline had a 3–6 fold increase in the soluble proline content of the leaf compared with the parent cultivar, Maris Mink. The mutant converted more [U-⁴C]glutamic acid to free proline in the leaves than Maris Mink but incorporation into protein proline was similar. Incorporation of radioactivity into proline was inhibited by exogenous proline more in Maris Mink than R5201, suggesting that feedback inhibition of proline biosynthesis is relaxed, but not absent in the mutant. When [1-¹⁴C]ornithine was the precursor, both R5201 and Maris Mink incorporated similar small amounts of label into soluble and protein proline. More protein proline was formed by both genotypes from labelled glutamic acid than from labelled ornithine. There may exist two routes of proline formation, where the glutamate pathway is synthetic and the ornithine pathway is catabolic.     

Effects of amino acid and trace element supplementation on pneumocandin production by Glarea lozoyensis: impact on titer, analogue levels, and the identification of new analogues of pneumocandin B0

Addition of the amino acids threonine, serine, proline, and arginine to fermentations of the fungus Glarea lozoyensis influenced both the pneumocandin titer and the spectrum of analogues produced. Addition of threonine or serine altered the levels of the “serine analogues” of pneumocandins B₀ and B₅ and allowed for their isolation and identification. Proline supplementation resulted in a dose-dependent increase in the levels of pneumocandins B₀ and E₀, whereas pneumocandins C₀ and D₀ decreased as a function of proline level. Moreover, proline supplementation resulted in an overall increase in the synthesis of both trans-3- and trans-4-hydroxyproline while maintaining a low trans-4-hydroxyproline to trans-3-hydroxyproline ratio compared to the unsupplemented culture. Pneumocandin production and the synthesis of hydroxyprolines was also affected by addition of the proline-related amino acid arginine but not by the addition of glutamine or ornithine. Zinc, cobalt, copper, and nickel, trace elements that are known to inhibit α-ketoglutarate-dependent dioxygenases, affected the pneumocandin B₀ titer and altered the levels of pneumocandins B₁, B₂, B₅, B₆, and E₀, analogues that possess altered proline, ornithine, and tyrosine hydroxylation patterns. Journal of Industrial Microbiology & Biotechnology (2001) 26, 216–221. Received 05 November 2000/ Accepted in revised form 27 January 2001     

Imino acid biosynthesis in Delonix regia

The biosynthesis of l-azetidine-2-carboxylic acid and trans-3-hydroxy-l-proline has been studied in Delonix regia seedlings by labelled precursor feeding techniques. α,γ-Diaminobutyric acid was incorporated into azetidine-2-carboxylic acid more efficiently than homoserine, methionine or aspartic acid. More radioactivity from proline was found in trans-3-hydroxyproline after 2 day's than after 4-day's metabolism, indicating a continuous turnover of the hydroxyimino acid in seedlings.     

Nouveaux acides amines libres de Afzelia bella: trans-hydroxy-4-L-proline et trans-carboxy-4-L-proline

Two new free amino acids have been isolated from seeds of Afzelia bella: trans-4-hydroxy-L-proline and trans-4-carboxy-L-proline. They are accompanied by relatively large quantities of proline, pipecolic, acid, 4-methylene-DL-proline, cis-4-hydroxymethyl-L-proline and 4-methylene-L-glutamic acid. Small amounts of acetylornithine are also present. It is the first time that trans-4-hydroxy-L-proline has been found as a component of the free amino pool of plants.     

Identification and Characterization of Bifunctional Proline Racemase/Hydroxyproline Epimerase from Archaea: Discrimination of Substrates and Molecular Evolution

Proline racemase (ProR) is a member of the pyridoxal 5’-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation) in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE), which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372) from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR). This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative) ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241) and natural ProR (phenylalanine). The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky) tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline), and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR.　A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily.     

Isosteric Phosphonate Pyrrolidine-Based Dinucleoside Monophosphate Analogues

Abstract

A novel α- and β-configured pyrrolidine nucleoside phosphonates in adenine series were synthesized from trans-4-hydroxy-L-proline as starting material. d(ApA) analogues were also prepared and studied with respect to their hybridization properties with polyU.     

Effect of novel compound, 1-methyl-1-piperidino methane sulfonate (MPMS), on the osmoprotectant activity of glycine betaine,cholien and l-proline in Escherichia coli

A novel compound, 1-methyl-1-piperidino methane sulfonate (MPMS), was found to block the osmoprotectant activity of choline and L-proline, but not glycine betaine in Escherichia coli. MPMS was more active against salt-sensitive than salt-resistant strains, but had no effect on the salt tolerance of a mutant which was unable to transport choline, glycine betaine and proline. Growth of E. coli in NaCl was inhibited by MPMS and restored by glycine betaine, but not by choline or L-proline. Uptake of radiolabeled glycine betaine, choline or L-proline by cells grown at high osmolarity was not inhibited when MPMS and the radioactive substrates were added simultaneously. Preincubation for 5 min with MPMS reduced the uptake of choline and L-proline, but not glycine betaine. Similar incubation with MPMS had no effect on the uptake of radiolabeled glucose or succinate. The toxicity of MPMS was much lower than that of the L-proline analogues L-azetidine-2-carboxylic acid and 3,4-dehydro-DL-proline. The exact mechanism by which MPMS exerts its effect is not entirely clear. MPMS or a metabolite may interfere with the activity of several independent permeases involved in the uptake of osmoprotective compounds, or the conversion of choline to glycine betaine, or effect the expression of some of the osmoregulatory genes.Abbreviations MPMS 1-methyl-1-piperidino-methane sulfonate     

A criterion to determine whether cis-4-hydroxyproline is produced in animal tissues

Hydrolyzates of tissues that had been labeled with [¹⁴C]proline often contain significant amounts of cis-4-hydroxy[¹⁴C]proline. Since animal cells do not contain an enzyme which can effect formation of cis-4-hydroxyproline, there are only two possible explanations for its presence. Either it is formed during acid hydrolysis of trans-4-hydroxyproline (which is synthesized by cells and is a common constituent of connective tissues), or it is produced by a nonenzymatic mechanism such as attack by oxygen radicals. It is important to resolve this issue because if a nonenzymatic mechanism is active in connective tissues, then it will be necessary to reevaluate currently accepted ideas about production of hydroxyproline. This communication describes a method for distinguishing between the two alternate explanations. Tissues or cells are labeled with [¹⁴C]proline, and then a known amount of trans-4-hydroxy[³H]proline is added to each sample before hydrolysis; the relative amounts of [¹⁴C]- and [³H]-cis-4-hydroxyproline are compared after hydrolysis. It is known from a separate series of measurements with mixtures of [¹⁴C]- and [³H]-trans-4-hydroxyproline standards that there is a very high correlation (r = 0.998) between acid-induced formation of the [¹⁴C]- and [³H]-cis epimers. One can thus compare the amount of cis-4-hydroxy[¹⁴C]proline in a hydrolyzate from a biological system with the amount that would be expected if it were all formed during acid hydrolysis. This method was used to show that fibroblasts cultured under conditions commonly used to study collagen metabolism do not produce cis-4-hydroxyproline. This result strongly suggests that nonenzymatic hydroxylation does not normally occur in cell culture systems.     

Effects of hydroxyproline on the growth and cell-wall protein metabolism of excised root segments of Pisum sativum

Summary Hydroxyproline, in the presence of sucrose, enhanced the extension growth of excised 2–4 mm pea root segments in aseptic media. About 90% of protein-bound hydroxyproline in the pea root segments was confined to the cell-wall fraction where it occurred as trans-4-hydroxy-l-proline. The amounts of wall-bound hydroxyproline increased dramatically towards the cessation of extension growth, but when the segments were cultured in trans-hydroxyproline, this increase was considerably less.Externally supplied cis and trans-hydroxyproline inhibited the formation of protein-bound [¹⁴C]hydroxyproline from [¹⁴C]proline without affecting the total amount of [¹⁴C]proline incorporated into proteins. Studies with -dipyridyl showed that, although some of the externally supplied trans-[¹⁴C]hydroxyproline was incorporated directly into cell-wall proteins, most of it was first converted into proline which was then incorporated into proteins and subsequently reconverted, in part, into hydroxyproline. The effect of externally supplied hydroxyproline is discussed in relation to protein-bound proline hydroxylation.     

D-Proline Effect on L-Proline-Requiring Mutants in Zea mays (L.)

D-Proline and three proline analogs, L-hydroxyproline, L-azetidine-2-carboxylicacid and L-thiazolidine-4-carboxylic acid, were tested for theireffect on proline-requiring mutants (pro 1) of maize at theconcentrations used for phenotypic repair with L-proline. D-Prolinewas the only one that promoted pro 1 mutant growth and did notaffect the growth of normal siblings. The possible role of D-prolinein repairing pro 1 mutants is discussed. (Received February 15, 1985; Accepted July 2, 1985)     

Influence of Proline and Glycinebetaine on Salt Tolerance of Cultured Barley Embryos

Lone, M. I., Kueh, J. S. H., Wyn Jones, R. G. and Bright, S.W. J. 1987. Influence of proline and glycinebetaine on salttolerance of cultured barley embryos.—J. exp. Bot. 38:479–490. The addition of exogenous proline and glycinebetaine to culturedbarley (Hordeum vulgare L. cv. Maris Mink) embryos increasedshoot elongation under saline conditions. Inhibition of shootelongation by NaCl was relieved by proline when plantlets weregrown in deep crystallizing dishes but not in Petri dishes whereshoots come into direct contact with the medium. The effectof proline could be related to a decrease in shoot Cl^–and Na⁺ accumulation which was only observed in plantlets grownin crystallizing dishes. Proline but not betaine uptake intocultured plantlets was stimulated by NaCl while each organicsolute inhibited the endogenous synthesis of the other soluteunder salt stress. Comparison of the effects of exogenously supplied proline withenhanced endogenous proline accumulation in the mutant lineR5201 suggested that the increased proline accumulation in themutant is an order of magnitude too low to have a significantphysiological effect. The implications of the effect of prolineon ion transport, discrimination and accumulation are discussed. Key words: Salt tolerance, proline, ion transport, barley embryo culture     

Identification of rapidly labeled, small molecular weight hydroxyproline-containing peptides in developing mouse limbs in vitro and in vivo

Limbs from embryonic mice labeled with radioactive proline either in vitro or in vivo readily synthesized hydroxyproline-containing peptides, the majority of which were of a molecular size less than intact collagen α-chains. The hydroxyproline isomers detected in these peptides included trans-3-hydroxyproline, trans-4-hydroxyproline, and cis-4-hydroxyproline. The abundance of small peptide material containing these hydroxyproline isomers is unusual in that as much as 85% is present in such forms at 10 days gestation when the limb is initially forming and 35% at 14 days when the limb is fully developed. The identification of small molecular weight hydroxyproline-containing peptides in limbs removed from embryos labeled in vivo indicate they are not organ culture artifacts.     

Identification of a human trans-3-hydroxy-L-proline dehydratase, the first characterized member of a novel family of proline racemase-like enzymes

A family of eukaryotic proline racemase-like genes has recently been identified. Several members of this family have been well characterized and are known to catalyze the racemization of free proline or trans-4-hydroxyproline. However, the majority of eukaryotic proline racemase-like proteins, including a human protein called C14orf149, lack a specific cysteine residue that is known to be critical for racemase activity. Instead, these proteins invariably contain a threonine residue at this position. The function of these enzymes has remained unresolved until now. In this study, we demonstrate that three enzymes of this type, including human C14orf149, catalyze the dehydration of trans-3-hydroxy-L-proline to Δ(1)-pyrroline-2-carboxylate (Pyr2C). These are the first enzymes of this subclass of proline racemase-like genes for which the enzymatic activity has been resolved. C14orf149 is also the first human enzyme that acts on trans-3-hydroxy-L-proline. Interestingly, a mutant enzyme in which the threonine in the active site is mutated back into cysteine regained 3-hydroxyproline epimerase activity. This result suggests that the enzymatic activity of these enzymes is dictated by a single residue. Presumably, human C14orf149 serves to degrade trans-3-hydroxy-L-proline from the diet and originating from the degradation of proteins that contain this amino acid, such as collagen IV, which is an important structural component of basement membrane.     

Mutant cell lines resistant to azetidine carboxylic acid: Quantitative and qualitative differences in pyrroline-5-carboxylate synthase activity

Mutant Chinese hamster lung fibroblasts were selected that are resistant to the proline analog L-azetidine-2-carboxylic acid. Resistance in the two mutant cell lines is associated with two distinct alterations in pyrroline-5-carboxylate synthase, the enzyme that catalyzes the proline biosynthetic step leading from glutamic acid to pyrroline-5-carboxylate. In one mutant cell line, pyrroline-5-carboxylate synthase specific activity is increased 30-fold over the level in control cells. In the other mutant line, pyrroline-5-carboxylate synthase activity is not increased, but the enzyme has become insensitive to inhibition by ornithine and proline.     

The metabolism of exogenous hydroxyproline by gametophytes of Plagiochila arctica Bryhn et Kaal. (Hepaticae)

The metabolic fate of free 4-hydroxyproline (Hyp), dual-labeled in specific positions, supplied to axenically cultured gametophytes of Plagiochila arctica was found to be complex. It could enter into at least three pathways, and the one that predominated depended on the concentration supplied. At low concentrations (i.e., 1 M), free Hyp was mostly converted to proline (Pro) by a pathway in which pyrroline-5-carboxylate is the most probable intermediate. Lesser amounts entered a pathway(s) in which hydrogen was lost from the 3(2) carbon. And a small amount was directly incorporated into protein. At the higher concentrations that have been found to desuppress leaf and branch development in P. arctica the proportion of the labeled Hyp converted to Pro was markedly diminished and the proportion that was directly incorporated into peptide linkage was increased. This direct incorporation of Hyp into protein may be closely related to the ability of Hyp to relieve suppression of ventral leaves in leafy liverworts. the suppression-desuppression phenomenon is thought to involve cell surface and-or extracellular matrix proteins.Abbreviations Hyp 4-trans-hydroxyl-L-proline - Pro L-proline     

Evidence for a role for L-proline as a salinity protectant in the cyanobacterium Nostoc muscorum

Nostoc muscorum required an active proline oxidase in order to assimilate exogenous proline as a source of fixed nitrogen. A mutant strain (Ac^r) resistant to growth inhibition by L-azetidine-2-carboxylate (AC) was found to be deficient in proline oxidase activity, and to be a proline overaccumulator. Proline overaccumulation, resulting either from mutational acquisition of the Ac^r phenotype or from salinity-inducible uptake of exogenous proline, conferred enhanced salinity tolerance in this cyanobacterium.     

Characterization and regeneration of salt- and water-stress mutants from protoplast culture of Nicotiana plumbaginifolia (Viviani)

Summary Protoplast-derived colonies of haploid N. plumbaginifolia leaves were used to select for resistance to NaCl, KCl and polyethylene glycol 6000 (PEG). Salt-and PEG-tolerant cell lines were isolated on the basis of growth in a culture medium containing inhibitory concentrations of either NaCl or KCl (200 mM) or PEG (25%). The frequency of resistant lines ranged from 10^-5 to 10^-6. One resistant line from each treatment was regenerated into plants. All resistant lines produced 10–25 times more proline than the wild type when grown on a non-selective medium. Similar values were also observed in the leaves of resistant progeny plants. In each mutant line, salt or PEG resistance was transmitted as a single dominant nuclear gene as shown by segregation ratios in progenies of crosses between resistant and wild-type plants. The latter observation demonstrates clearly the existence of a genetic basis for increased salt tolerance.     

Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination,callus and shoot formation

A novel acetyltransferase (Mpr1) found in Saccharomyces cerevisiae (strain 1278b) has been shown to specifically detoxify a proline analog, l-azetidine-2-carboxylic acid (A2C) in yeast cells [M. Shichiri et al. (2001) J Biol Chem 276: 41998–42002]. We investigated whether the yeast MPR1 gene would function similarly in a plant system and if its expression could confer resistance to proline analogs. The MPR1 gene coding sequence driven by two different constitutive promoters, with or without the 5- and 3-noncoding sequence from the MPR1 gene adjacent to the conventional NOS terminator, was transformed into tobacco (Nicotiana tabacum L. cv. Xanthi) plants via Agrobacterium tumefaciens infection. The presence of the yeast 5- and 3-noncoding sequences appeared to increase the likelihood of MPR1 gene expression in the transgenic plants. The kanamycin-selected transgenic plants with a high level of Mpr1 activity grew normally, and their progeny expressed acetyltransferase activity that could utilize A2C, azetidine-3-carboxylic acid and 4-hydroxy-l-proline as substrates. Resistance to A2C, but not to the other two analogs, was exhibited during leaf tissue culture and seed germination. The A2C toxicity to the wild-type plants was reversed by the addition of proline, suggesting that A2C acts as a proline analog. Our studies confirm that MPR1 can function in a similar fashion in tobacco as in yeast to detoxify the toxic proline analog A2C, so it could potentially be used as a new selectable marker for plant transformation. However, our attempts to utilize MPR1 as an efficient selectable marker gene for the A. tumefaciens-mediated transformation of tobacco were unsuccessful.Abbreviations A2C: l-Azetidine-2-carboxylic acid - A3C: Azetidine-3-carboxylic acid - Hyp: 4-Hydroxy-l-proline - hpt: Hygromycin phosphotransferase II - NPTII: Neomycin phosphotransferase II Communicated by H. Wang     

Mutant cell lines resistant to azetidine-2-carboxylic acid: alterations in the synthesis of proline from glutamic acid

Two mutant Chinese hamster lung fibroblast lines have been isolated that are resistant to the toxic proline analog L-azetidine-2-carboxylic acid. The line designated AZCA-1 has 30-fold elevated activity of pyrroline-5-carboxylate synthase and a large increase in the rate of proline production and release compared to controls. Pyrroline-5-carboxylate synthase activity is not elevated in the resistant line designated AZCA-4, but the enzyme is less sensitive to inhibition by ornithine and proline than control enzyme. Intracellular proline is elevated in AZCA-4 cells, with no change in the rate of release of proline synthesized from glutamate. Resistance to azetidine carboxylic acid in both mutant lines is attributed to the expanded intracellular proline pool that results from alterations in pyrroline-5-carboxylate synthase. These results indicate that intracellular proline levels are determined at least in part by the regulated activity of pyrroline-5-carboxylate synthase.     

Isolation and characterization of amino acid-analogue-resistant mutants of Spirulina platensis

Amino acid-analogue-resistant mutants of the cyanobacterium Spirulina platensis were isolated using amino acid analogues -2-thienylalanine, p-fluorophenylalanine, ethionine and azetidine-2-carboxylic acid. The growth and other cellular contents in these mutants were less than in the parent. The internal free amino acid pool showed varying amounts. Maximal overproduction occurred of proline whereas overproduction of aspartic acid, alanine and lysine was much less.