Action of a proline analogue, l-thiazolidine-4-carboxylic acid, in Escherichia coli

Unger, Leon (University of Illinois, Urbana), and R. D. DeMoss. Action of a proline analogue, l-thiazolidine-4-carboxylic acid, in Escherichia coli. J. Bacteriol. 91:1556-1563. 1966.-The effect of the proline analogue, l-thiazolidine-4-carboxylic acid (thioproline), on growth, and its relation to the metabolic function of proline in protein synthesis in Escherichia coli K-12, has been studied. Thioproline causes linear growth in E. coli within one generation. The inhibition is specifically reversed by the simultaneous addition of l-proline. Thioproline, or a closely related metabolic derivative, is incorporated into bacterial proteins. Proline antagonizes the incorporation of "thioproline" into protein. The analogue specifically inhibits the rate and extent of prolyl-ribonucleic acid formation. The effectiveness of thioproline as a proline analogue is attributed to its ability to interfere with the utilization of proline for protein synthesis and to mimic proline in its function of being incorporated into proteins. The effect of the incorporation of thioproline on protein structure and enzyme activity is discussed.     

Macrophage-mediated N-nitrosation of thioproline and proline

Thioproline (Thiazolidine-4-carboxylic acid) and proline were nitrosated by stimulated mouse macrophages in vitro. A macrophage cell line (J774.1, 1.0 x 10(6)/well, 1 ml) was incubated with Escherichia coli lipopolysaccharide, interferon-gamma and thioproline (5 mM) or proline (5 mM). After 72 hr incubation at 37 degrees C, 4 microM N-nitrosothioproline was produced. The amount of N-nitrosoproline was much lower than that of N-nitrosothioproline. Thioproline and proline inhibited the formation of carcinogenic N-nitrosomorpholine. N-nitrosothioproline and N-nitrosoproline are found as major N-nitroso compounds in human urine. Macrophage mediated N-nitrosation may contribute to the formation of these N-nitrosamino acids in the human body.     

Purification, properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum

1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.     

Effect of the proline analogue baikiain on proline metabolism in Salmonella typhimurium

A proline analogue, 4,5-dehydro-l-pipecolic acid (baikiain) induces the formation in Salmonella typhimurium of the two enzymes catalyzing the degradation of proline, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid (P5C) dehydrogenase. The level of induction by 20 mm baikiain is about 10% of the maximum level induced by proline. Since the analogue is a substrate of proline oxidase the first enzyme of the proline catabolic pathway, the oxidation derivative rather than baikiain itself might be the actual effector. Baikiain is also an inducer of proline oxidase in Escherichia coli K-12 and E. coli W. An additional effect of this analogue on proline degradation in S. typhimurium is inhibition of P5C dehydrogenase. At a concentration of 5 x 10(-4)m, baikiain inhibits completely the growth of strains constitutive for proline oxidase. This inhibition, which can be overcome by proline, occurs in the presence or absence of P5C dehydrogenase activity. Three spontaneously occurring mutants resistant to baikiain were isolated from constitutive strains. All are pleiotropic-negative for the proline-degrading enzymes. The sites of these mutations are linked to the put region. Although the mechanism of toxicity has not been determined, baikiain provides a simple and direct selection for obtaining mutants unable to degrade proline. In addition, it allows selection for strains with an inducible rather than constitutive phenotype.     

Thioproline-induced accumulation of proline in barley leaf sections

Abstract Treatment of barley leaf sections with 0.1 mol m^?3 thioproline (L-thiazolidine-4-carboxylic acid) was found to induce a marked increase in proline together with some decrease in glutamate, whereas the levels of other andno acids were not influenced. This result is discussed in relation to the significance of the increase in proline in tissues treated with abscisic acid or subjected to water stress.     

Action of thiazolidine-2-carboxylic acid, a proline analog, on protein synthesizing systems.

Thiazolidine-2-carboxylic acid, or beta-thiaproline, is a proline analog in which the beta methylene group of proline is substituted by a sulfur atom. It has been deomonstrated that beta-thiaproline is activated and transferred to tRNAPro by Escherichia coli and rat liver aminoacyl-tRNA synthetases, and inhibits proline incorporation into polypeptides in protein synthesizing systems from E. coli, rat liver or rabbit reticulocytes. In mammalian systems beta-thiaproline inhibits also leucine incorporation; in rabbit reticulocyte lysate it inhibits ribosome run-off. Both these effects may be explained by the fact that beta-thiaproline once incorporated into the growing polypeptide chain impairs its further elongation, as shown by experiments made with puromycin. All tests were performed in comparison with thiazolidine-4-carboxylic acid, or gamma-thiaproline, another proline analog having the gamma methylene group substituted by a sulfur atom; it was shown that in all the reactions studied both compounds act as competitive inhibitors of proline. Some differences in the effects of the two analogs have been evidenced: in almost all the reactions and mainly in the whole protein synthesizing systems, beta-thiaproline shows an higher inhibitory activity.     

The effects of proline, thioproline and methylglyoxal-bis-(guanylhydrazone) on shoot regeneration frequencies from stem explants of B. napus

Internode segments from aseptic shoot cultures are the most prolific explants for the regeneration of Brassica shoots in vitro. These explants also have the advantage of not being subject to the genotypic variations in regeneration response observed in hypocotyl and cotyledon explants. Despite reports of 80–100% shoot regeneration from stem explants, observed frequencies are typically 50–60%. Three media additives, proline, thioproline and methylglyoxal-bis-(guanylhydrazone) (MGBG), were tested for their efficacy in promoting shoot regeneration from stem explants of two B. napus varieties, Westar and Cobra. The effects of proline and thioproline on both varieties were neutral or deleterious. In Cobra the MGBG treatments caused a uniform reduction in explant regeneration. However, at low concentrations (0.35M) MGBG resulted in a 50% increase, to 92%, in regeneration from Westar. The potential of MGBG in promoting explant regeneration in B. napus is discussed in the light of its interaction with the explant genotype.Abbreviations ABA abscisic acid - BAP benzylaminopurine - MGBG methylglyoxal-bis-(guanylhydrazone) - NAA naphthalene acetic acid - thioproline thiazolidine-4-carboxylic acid     

Synthesis of the proline analogue [2,3-3H]azetidine-2-carboxylic acid. Uptake and incorporation in Arabidopsis thaliana and Escherichia coli.

Azetidine-2-carboxylic acid, the 4-membered ring noranalogue of proline, is regularly used in the study of proline metabolism as well as the study of protein conformation. We prepared D,L-[2,3-3H]azetidine-2-carboxylic acid with an optimized 10% yield from commercially available 4-amino-[2,3-3H]butyric acid. Purification was performed by fast-protein liquid chromatography. The biological activity was checked in both Arabidopsis thaliana and Escherichia coli. The obtained specific activity of 10 mCi/mmol was sufficient for most uptake and incorporation studies.     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

Isolation, properties and tissue distribution of rat glutathione transferase E

Uptake of Li+ induced by the addition of proline to a cell suspension of Escherichia coli was detected using an Li+-selective electrode. This Li+ uptake was inhibited by L-azetidine 2-carboxylic acid, a competitive inhibitor of the proline transport system. Thus, direct evidence for Li+-proline cotransport via the proline transport system was obtained. Kinetic parameters of the Li+ uptake were determined.     

Site-specific alteration of cysteine 281, cysteine 344, and cysteine 349 in the proline carrier of Escherichia coli

Cys-281, Cys-344, or Cys-349 in the proline carrier of Escherichia coli was changed to a serine residue by site-specific mutagenesis. The activities of the resultant mutants for uptake of proline were as great as that of the wild-type strain. These mutant carriers were all as sensitive as the wild-type carrier to the proline analogue azetidine 2-carboxylate. However, the mutant carriers with Ser-281 and Ser-344 were resistant to N-ethylmaleimide, whereas the mutant carrier with Ser-349 was as sensitive as the wild-type carrier to this reagent. These results indicate that these cysteine residues are not essential for proline transport and that Cys-281 and Cys-344 may be close to the substrate-binding site that contains an N-ethylmaleimide-sensitive residue.     

Regulation of Proline Degradation in Salmonella typhimurium

The pathway for proline degradation in Salmonella typhimurium appears to be identical to that found in Escherichia coli and Bacillus subtilis. Delta(1)-Pyrroline-5-carboxylic acid (P5C) is an intermediate in the pathway; its formation consumes molecular oxygen. Assays were devised for proline oxidase and the nicotinamide adenine dinucleotide phosphate-specific P5C dehydrogenase activities. Both proline-degrading enzymes, proline oxidase and P5C dehydrogenase, are induced by proline and are subject to catabolite repression. Three types of mutants were isolated in which both enzymes are affected: constitutive mutants, mutants with reduced levels of enzyme activity, and mutants unable to produce either enzyme. Most of the mutants isolated for their lack of P5C dehydrogenase activity have a reduced level of proline oxidase activity. All the mutations are cotransducible. A genetic map of some of the mutations is presented. The actual effector of the pathway appears to be proline.     

The x ray structure of thiazolidine-4-carboxylic acid

The X ray crystal structure of thiazolidine-4-carboxylic acid has been determined. It appears that the anomalously low basicity of this compound in comparison with that of its non-sulfur-containing analogue, proline, is due to the resonance stabilization of the unprotonated form of thiazolidine-4-carboxylic acid. This stabilization is conferred through an increased interaction of the sulfur atom with the thiazolidine-4-carboxylic acid ring that is only possible when the nitrogen atom maintains a trigonal geometry. This effect seems also to account for the greater instability of N-hydroxymethylamines formed from thiazolidine-4-carboxylic acid as compared to those formed from proline. The crystal structure shows, in addition, an intramolecular hydrogen bond in combination with a bifurcated hydrogen bond.     

Coformational aspects of polypeptide structure XL. The synthesis of poly((S)-thiazolidine-4-carboxylic acid)

The synthesis of poly[(S)-thiazolidine-4-carboxylic acid] is described. The polymer is obtained by the polymerization of the N-carboxyanhydride of (S)-thiazolidine-4-carboxylic acid in pyridine or nitrobenzene using triethylamine as an initiator. The amino acid is prepared by the condensation of cysteine and formaldehyde. N-Acetyl-(S)-thiazolidine-4-carboxylic acid methyl ester is also prepared as a model compound by standard acetylation and esterification reactions.     

The in vivo inhibition of collagen synthesis and the reduction of prolyl hydroxylase activity by 3,4-dehydroproline.

Various proline analogs and iron chelators were tested for their effect on collagen formation which occurs in the uterus of the immature rat following the administration of estradiol-17β. dl-3,4-Dehydroproline, l-α-azetidine-2-carboxylic acid and l-pyroglutamic acid reduced the estradiol-17β stimulated formation of hydroxyproline which occurs in the uterus following administration of the hormone while l-thiazolidine-4-carboxylic acid was without effect on this response. The activity of the d- and l-isomers of 3,4-dehydroproline was compared with the racemic mixture; the l-isomer was twice as active as the latter, while the d-isomer was only half as active. l-3,4-Dehydroproline was approximately four times as potent as l-α-azetidine-2-carboxylic acid, the second most active analog of those tested. dl-3,4-Dehydroproline inhibited the incorporation of l-[¹⁴C]proline into the proline and hydroxyproline of uterine collagen; it also inhibited the incorporation of [¹⁴C]glycine into collagen while having less effect on the incorporation of these amino acids into noncollagen protein. These results indicate dl-3,4-dehydroproline is a fairly specific and potent inhibitor of collagen formation in vivo.These observations indicate that dl-3,4-dehydroproline reduces the hydroxylation of prolyl residues in collagen. Presumably, this occurs in part due to the incorporation of the analog into the collagen molecule in place of proline. It is probably also related to a reduction of prolyl hydroxylase activity which can be demonstrated in the tissues of animals treated with 3,4-dehydroproline. A significant reduction of prolyl hydroxylase activity was shown to persist in the uterus, lung, and heart for approximately 24 h following a single intraperitoneal dose of dl-3,4-dehydroproline (200 mg/kg).     

Mutations in the listerial proB gene leading to proline overproduction: effects on salt tolerance and murine infection

The observed sensitivity of Listeria monocytogenes to the toxic proline analogue L-azetidine-2-carboxylic acid (AZ) suggested that proline synthesis in Listeria may be regulated by feedback inhibition of gamma-glutamyl kinase (GK), the first enzyme of the proline biosynthesis pathway, encoded by the proB gene. Taking advantage of the Epicurian coli mutator strain XL1-Red, we performed random mutagenesis of the recently described proBA operon and generated three independent mutations in the listerial proB homologue, leading to proline overproduction and salt tolerance when expressed in an E. coli (DeltaproBA) background. While each of the mutations (located within a conserved 26-amino-acid region of GK) was shown to confer AZ resistance (AZ(r)) on an L. monocytogenes proBA mutant, listerial transformants failed to exhibit the salt-tolerant phenotype observed in E. coli. Since proline accumulation has previously been linked to the virulence potential of a number of pathogenic bacteria, we analyzed the effect of proline overproduction on Listeria pathogenesis. However, our results suggest that as previously described for proline auxotrophy, proline hyperproduction has no apparent impact on the virulence potential of Listeria.     

Effects of the Proline Analog l-Thiazolidine-4-carboxylic Acid on Proline Metabolism

The effect of various proline analogs on proline oxidation in mitochondria isolated from etiolated barley (Hordeum vulgare) shoots was investigated. Of the analogs tested, only l-thiazolidine-4-carboxylic acid (T4C) was an effective inhibitor. T4C (1 millimolar) inhibited proline (10 millimolar) -dependent 0₂ uptake an average of 67%. T4C was also oxidized to some degree (12.9 nanoatoms oxygen per minute per milligram protein for 10 millimolar). The effect of T4C on the oxidation of other mitochondrial substrates was also tested. T4C inhibited ¹-pyrrolidine-5-carboxylic acid-dependent oxygen uptake slightly (13%), the oxidation of malate plus pyruvate even less (6%), and stimulated the oxidation of succinate (+11%), exogenous NADH (+19%), and citrate (+20%). Thus, inhibition by T4C in mitochondria is relatively specific to proline oxidation. T4C was found to inhibit proline dehydrogenase and not the transport of proline into the matrix.     

Nutritional and metabolic interrelationships of arginine, glutamic acid and proline in the chicken.

Proline satisfies by a narrow margin the criterion for dietary essentially for the chick. It is estimated that the chick may synthesize 80-90% of the total proline needed for growth. Although the metabolism of arginine, ornithine and glutamic acid is expected to give rise to proline, dietary supplements to these amino acids are relatively ineffective in reducing the proline requirement of chicks. Studies of the efficacy of dietary ornithine for growth, and tracer studies using L-(5-3H)arginine indicate that the conversion of ornithine to proline in vivo is limited, and the amount of proline synthesized from arginine is but a small fraction of that needed for growth. The limiting processes in proline synthesis from glutamic acid and ornithine are not known. In Escherichia coli, where the biosynthetic pathway from glutamate to proline has been elucidated, a glutamate kinase, NADP-dependent delta1-pyrroline-5-carboxylic acid (P5C) dehydrogenase and P5C reductase catalyze proline synthesis. P5C reductase is present in the soluble fraction of chicken liver and kidney. An NADP-dependent P5C dehydrogenase activity has also been observed in this fraction of liver. Further studies are required to assess the importance of these enzymes in proline biosynthesis and to determine the limiting process in proline formation in the chicken.     

Antibacterial activity of phosphono peptides based on 4-amino-4-phosphonobutyric acid

Abstract Phosphono dipeptides based on 4-amino-4-phosphonobutyric acid (phosphonic acid analogue of glutamic acid, GluP) were synthesized and evaluated for their antibacterial activity. Dipeptides containing N-terminal alanine, leucine, isoleucine, phenylalanine or lysine showed marked antibacterial activity against Escherichia coli , whilst those containing alanine, leucine, valine or proline were active against Serratia marcescens . AlaGluP and LeuGluP were nearly equipotent with the respective dipeptides based on 1-aminoethylphosphonic acid (phosphonic acid analogue of alanine). The structure-activity relationship, i.e. dependence of the activity of phosphono dipeptides on the nature of their N-terminal component, indicated that transport of the peptide through the bacterial cytoplasmic membrane constitutes a crucial step in its antibacterial activity.