Proline Oxidase Research Articles

Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae

We examined the role of intracellular proline under freezing and desiccation stress conditions in Saccharomyces cerevisiae. When cultured in liquid minimal medium, the proline-nonutilizing mutant containing the put1 mutation (proline oxidase-deficient) produced more intracellular proline, and increased the cell survival rate as compared to the wild-type strain after freezing and desiccation. We also constructed two PUT1 gene disruptants. PUT1-disrupted mutants in minimal medium supplemented with external proline at 0.1% accumulated higher proline levels than those of the control strains (17–22-fold). These disruptants also had a 2–5-fold increase in cell viability compared to the control strains after freezing and desiccation stresses. These results indicate that proline has a stress-protective function in yeast.

Arginine catabolism in the cyanobacterium Synechocystis sp. Strain PCC 6803 involves the urea cycle and arginase pathway.

Cells of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 supplemented with micromolar concentrations of L-[(14)C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of L-[(14)C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [(14)C]ornithine preceded that of [(14)C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with L-[(14)C]ornithine, suggesting that the reaction of arginase, rendering ornithine and urea, is the main initial step in arginine catabolism. Ornithine followed two metabolic pathways: (i) conversion into citrulline, catalyzed by ornithine carbamoyltransferase, and then, with incorporation of aspartate, conversion into argininosuccinate, in a sort of urea cycle, and (ii) a sort of arginase pathway rendering glutamate (and glutamine) via Delta(1)pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an argF (ornithine carbamoyltransferase) insertional mutant was impaired in the production of [(14)C]citrulline from [(14)C]arginine; (ii) a proC (Delta(1)pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [(14)C]proline, [(14)C]glutamate, and [(14)C]glutamine from [(14)C]arginine or [(14)C]ornithine; and (iii) a putA (proline oxidase) insertional mutant did not produce [(14)C]glutamate from L-[(14)C]arginine, L-[(14)C]ornithine, or L-[(14)C]proline. Mutation of two open reading frames (sll0228 and sll1077) putatively encoding proteins homologous to arginase indicated, however, that none of these proteins was responsible for the arginase activity detected in this cyanobacterium, and mutation of argD (N-acetylornithine aminotransferase) suggested that this transaminase is not important in the production of Delta(1)pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [(14)C]arginine catabolism also provide a rationale for understanding how nitrogen is made available to the cell after mobilization of cyanophycin [multi-L-arginyl-poly(L-aspartic acid)], a reserve material unique to cyanobacteria.

Neurotransmitter Levels and Synaptic Strength at the Drosophila Larval Neuromuscular Junction are not Altered by Mutation in the Sluggish-a Gene, Which Encodes Proline Oxidase and Affects Adult Locomotion

The sluggish-A (slgA) gene of Drosophila melanogaster has been shown to encode for the enzyme proline oxidase, a mitochondrial enzyme which catalyzes the first step in the conversion of L-proline to L-glutamate. The slgA transcript is expressed in both larval and adult Drosophila melanogaster. Mutations in this gene lead to reduced proline oxidase activity and an elevation of free proline levels. Adult mutant flies show a striking reduction of motor activity. Since proline oxidase may contribute to the supply of the neurotransmitter glutamate in the nervous system, a reduction in proline oxidase activity could reduce neural glutamate pools and affect synaptic transmission in neurons utilizing glutamate as a transmitter, including peripheral motor neurons. We tested the hypothesis that glutamate, and synaptic transmission mediated by glutamate, are reduced at synapses of glutamatergic motor neurons in slgA mutants. Levels of glutamate and proline in different cell compartments, and functional properties of synaptic transmission were compared in slgA and control specimens. Proline is elevated in muscle cells of slgA mutants, indicating that the slgA gene regulates tissue proline levels. In nerve terminal varicosities, proline levels were low in both mutants and controls. Glutamate levels in nerve terminal varicosities of slgA mutants and controls were similar. In addition, we found that glutamatergic synaptic transmission at individual nerve endings and at the whole-cell level was similar in slgA mutants and controls. Thus, proline oxidase does not play a major role in generating neuronal glutamate pools at the Drosophila larval neuromuscular junction, and larval neuromuscular performance is not altered significantly in slgA mutants. Metabolic pathways other than that involving proline oxidase are able to sustain glutamatergic synaptic function in Drosophila larvae.

Proline Metabolism Under Water Stress in the Leaves and Roots of Brassica juncea Cultivars Differing in Drought Tolerance

Plant water relations, proline content and activities of pyrroline-5-carboxylate synthetase (P5CS) and proline oxidase (PO) were studied in five Brassica juncea genotypes differing in drought response. Under water stress, drought tolerant cv Varuna showed maximum osmotic adjustment value (1/b) of 3.21. In this variety, proline content increased from 8.4 and 5.3 μmol g−1 dry weight under normal conditions to 128.2 and 44.5 μmol g−1 dry weight under stress conditions in the leaves and roots respectively. The increase in proline content in less drought tolerant variety Prakash was from 7.0 and 1.8 μmol g−1 dry weight In the absence of stress to 62.9 and 13.4 μmol g−1 dry weight in the presence of stress in leaves and roots respectively. Compared to the respective controls, increase in the activity of proline biosynthetic enzyme P5CS of roots and leaves under water stress was maximum (∼ 94%) in Varuna and minimum in Prakash (∼16%). Reduction in the activity of proline degrading enzyme PO due to stress was also maximum in the leaves (75%) and roots (87%) of Varuna. These results showed genotype dependent effect of water stress on plant water relations and proline metabolism.

Effect of Mutations in Genes PH085 and PH04 on Proline Utilization in Yeast Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae can utilize proline as a single nitrogen source. We have found that the strains with mutations in gene PH085, a structural gene encoding cyclin-dependent phosphoprotein kinase, cannot grow on the proline-containing media. The ability to utilize proline is restored in strains of the pho85pho4genotype. We suggest that phosphoprotein kinase Pho85p is involved in either phosphorylation of a highly specific proline permease, Put4p, or in signaling proline concentration. The Pho4p protein that activates transcription of the PH05 gene, a structural gene of acid phosphatase, seems to participate in the negative regulation of the PUT1 and PUT2 genes encoding enzymes of proline catabolism, proline oxidase and Δ-pyrroline-5-carboxylate dehydrogenase. Thus, regulation of phosphorus and nitrogen metabolism have common elements.

Responses of Ceriops Roxburghiana to NaCl Stress

Responses of Ceriops roxburghiana Arn. leaves to the sodium chloride, applied at different concentrations (ranging from 100 to 600 mM), has been evaluated. Total amino acid content decreased with increasing NaCl concentration, while the protein content increased significantly up to 400 mM concentration and decreased thereafter. Total sugar content decreased at concentrations beyond 400 mM. Proline and glycine betaine were accumulated with increasing NaCl concentration. Protease and ATPase activities were increased whereas proline oxidase activity were decreased with increasing salinity. Peroxidase and malate dehydrogenase (NADH-MDH) activities did not significantly differ under various NaCl concentrations.

Lactate inhibits citrulline and arginine synthesis from proline in pig enterocytes.

Hypocitrullinemia and hypoargininemia but hyperprolinemia are associated with elevated plasma concentration of lactate in infants. Because the small intestine may be a major organ for initiating proline catabolism via proline oxidase in the body and is the major source of circulating citrulline and arginine in neonates, we hypothesized that lactate is an inhibitor of intestinal synthesis of citrulline and arginine from proline. To test this hypothesis, jejunum was obtained from 14-day-old suckling pigs for preparation of enterocyte mitochondria and metabolic studies. Mitochondria were used for measuring proline oxidase activity in the presence of 0-10 mM L-lactate. For metabolic studies, enterocytes were incubated at 37 degrees C for 30 min in Krebs bicarbonate buffer (pH 7.4) containing 5 mM D-glucose, 2 mM L-glutamine, 2 mM L-[U-14C]proline, and 0, 1, 5, or 10 mM L-lactate. Kinetics analysis revealed noncompetitive inhibition of intestinal proline oxidase by lactate (decreased maximal velocity and unaltered Michaelis constant). Lactate had no effect on either activities of other enzymes for arginine synthesis from proline or proline uptake by enterocytes but decreased the synthesis of ornithine, citrulline, and arginine from proline in a concentration-dependent manner. These results demonstrate that lactate decreased intestinal synthesis of citrulline and arginine from proline via an inhibition of proline oxidase and provide a biochemical basis for explaining hyperprolinemia, hypocitrullinemia, and hypoargininemia in infants with hyperlactacidemia.

The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria.

The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria.

Rice callus growth, proline content and activity of proline and IAA oxidase under the influence of hydroxyproline and NaCl

Calli of salt tolerant (Bhoora rata) and salt susceptible (GR11) rice varieties were cultured on Linsmaeir and Skoog’s medium containing LD50 concentration of NaCl (200 mM) and hydroxyproline (10 mM). Growth, proline content and activity of proline and IAA oxidases of the cultured tissues were determined at the end of 0, 2, 4, and 6 weeks of incubation. Hydroxyproline resistant calli of both rice varieties when cultured on Linsmaeir and Skoog’s medium containing hydroxyproline and NaCl showed increased dry weight and proline content as compared to NaCl stressed calli. The levels of proline and IAA oxidases were also low in the hydroxyproline resistant calli.

Endogenous Synthesis of Arginine Plays an Important Role in Maintaining Arginine Homeostasis in Postweaning Growing Pigs

This study was conducted to determine whether endogenous synthesis of arginine plays a role in regulating arginine homeostasis in postweaning pigs. Pigs were fed a sorghum-based diet containing 0.98% arginine and were used for studies at 75 d of age (28.4 kg body weight). Mitochondria were prepared from the jejunum and other major tissues for measuring the activities of 1-pyrroline-5-carboxylate (P5C) synthase and proline oxidase (enzymes catalyzing P5C synthesis from glutamate and proline, respectively) and of ornithiΔne aminotransferase (OAT) (the enzyme catalyzing the interconversion of P5C into ornithine). For metabolic studies, jejunal enterocytes were incubated at 37°C for 30 min in Krebs-Henseleit bicarbonate buffer containing 2 mmol/L L-glutamine, 2 mmol/L L-[U-14C]proline, and 0–200 μmol/L gabaculine (an inhibitor of OAT). The activities of P5C synthase, proline oxidase and OAT were greatest in enterocytes among all of the tissues studied. Incubation of enterocytes with gabaculine resulted in decreases (P< 0.05) in the synthesis of ornithine and citrulline from glutamine and proline. When gabaculine was orally administered to pigs (0.83 mg/kg body weight) to inhibit intestinal synthesis of citrulline from glutamine and proline, plasma concentrations of citrulline (−26%) and arginine (−22%) decreased (P< 0.05), whereas those of alanine (+21%), ornithine (+17%), proline (+107%), taurine (+56%) and branched-chain amino acids (+21–40%) increased (P< 0.05). On the basis of dietary arginine intake and estimated arginine utilization, the endogenous synthesis of arginine in the 28-kg pig provided ≥50.2% of total daily arginine requirement. Taken together, our results suggest an important role for endogenous synthesis of arginine in regulating arginine homeostasis in postweaning growing pigs, as previously shown in neonatal pigs.

Synthesis of citrulline and arginine from proline in enterocytes of postnatal pigs.

The synthesis of ornithine, citrulline, and arginine from proline was quantified in enterocytes of newborn (0-day-old) pigs, 2- to 21-day-old suckling pigs, and 29- to 58-day-old pigs weaned at 21 days of age. Mitochondria were prepared from enterocytes for measurement of proline oxidase activity. For metabolic studies, cells were incubated at 37 degrees C for 30 min in Krebs bicarbonate buffer (pH 7.4) containing 5 mM D-glucose and 0.0-5.0 mM L-[U-14C]proline with or without 2.0 mM L-glutamine. Proline oxidase activity and rates of synthesis of citrulline and arginine from proline were high in enterocytes of newborn pigs and markedly decreased in 7-day-old pigs. With increasing piglet age from 7 to 21 days, enterocyte proline oxidase activity and rate of conversion of proline into citrulline progressively increased, but the values in 21-day-old pigs remained much lower than in newborn pigs. The synthesis of ornithine, citrulline, and arginine from 0.5-5 mM proline was concentration dependent and required the addition of glutamine. About 80-90% of utilized proline carbons were recovered in ornithine plus citrulline plus arginine, with CO2 being a minor product. These results demonstrate a hitherto unrecognized pathway for the synthesis of ornithine, citrulline, and arginine via proline oxidase in enterocytes and challenge the current concept that pyrroline-5-carboxylate (P5C) is synthesized in the small intestine only from glutamine/glutamate via P5C synthase on the basis of previous work with postnatal rats.

Cloning and characterization of cDNAs encoding human proline oxidase, the enzyme deficient in type I hyperprolinemia. † 615

Cloning and characterization of cDNAs encoding human proline oxidase, the enzyme deficient in type I hyperprolinemia. † 615

Amelioration of NaCl stress by triadimefon in peanut seedlings

Peanut (Arachis hypogaea L.cv. VRI-2) seeds were imbibed in distilled water(control), 30 mM NaCl (stressed) and 30 mM NaCl + 10 mg L-1 triadimefon for 12 h and grown in a seed germinator with a day/night temperature of 30/22 °C and a photoperiod of 16 h with a PPFD of 250 µEm-2 s-1. Seedlings were irrigated with respective treatment solutions. Fifteen days old seedlings were harvested and used for the study. The NaCl stress decreased seedling growth, dry weight, photosynthetic pigments, protein content and the level of proline oxidase activity and it increased proline, glycine betaine content, protease and ATPase activities. Triadimefon treatment ameliorated the stress caused NaCl by increasing root growth, dry weight, chlorophyll, carotenoid, protein and glycine betaine contents and by decreasing proline accumulation, proline oxidase levels and ATPase. The stress amelioration conferred by triadimefon may be mediated by increased protein synthesis, osmoregulation and reduced energy requirement.

Reciprocal regulation of delta 1-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants.

Plants generally accumulate free proline under osmotic stress conditions. Upon removal of the osmotic stress, the proline levels return to normal. In order to understand the mechanisms involved in regulating the levels of proline, we cloned and characterized a proline dehydrogenase (PDH) cDNA from Arabidopsis thaliana (AtPDH). The 1745 bp cDNA contains a major open reading frame encoding a peptide of 499 amino acids. The deduced amino acid sequence has high homology with both Saccharomyces cerevisiae and Drosophila melanogaster proline oxidases and contains a putative mitochondrial targeting sequence. When expressed in yeast, the AtPDH cDNA complemented a yeast put1 mutation and exhibited proline oxidase activity. We also determined the free proline contents and the delta 1-pyrroline-5-carboxylate synthetase (P5CS) and PDH mRNA levels under different osmotic stress and recovery conditions. The results demonstrated that the removal of free proline during the recovery from salinity or dehydration stress involves an induction of the PDH gene while the activity of P5CS declines. The reciprocal regulation of P5CS and PDH genes appears to be a key mechanism in the control of the levels of proline during and after osmotic stress. The PDH gene was also significantly induced by exogenously applied proline. The induction of PDH by proline, however, was inhibited by salt stress.

Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator.

In many plants, osmotic stress induces a rapid accumulation of proline through de novo synthesis from glutamate. This response is thought to play a pivotal role in osmotic stress tolerance [Kishor, P. B. K., Hong, Z., Miao, G.-H., Hu, C.-A. A. and Verma, D. P. S. (1995) Plant Physiol. 108, 1387-1394]. During recovery from osmotic stress, accumulated proline is rapidly oxidized to glutamate and the first step of this process is catalyzed by proline oxidase. We have isolated a full-length cDNA from Arabidopsis thaliana, At-POX, which maps to a single locus on chromosome 3 and that encodes a predicted polypeptide of 499 amino acids showing significant similarity with proline oxidase sequences from Drosophila and Saccharomyces cerevisiae (55.5% and 45.1%, respectively). The predicted location of the encoded polypeptide is the inner mitochondrial membrane. RNA gel blot analysis revealed that At-POX mRNA levels declined rapidly upon osmotic stress and this decline preceded proline accumulation. On the other hand, At-POX mRNA levels rapidly increased during recovery. Free proline, exogenously added to plants, was found to be an effective inducer of At-POX expression; indeed, At-POX was highly expressed in flowers and mature seeds where the proline level is higher relative to other organs of Arabidopsis. Our results indicate that stress- and developmentally derived signals interact to determine proline homeostasis in Arabidopsis.

A nuclear gene encoding mitochondrial proline dehydrogenase, an enzyme involved in proline metabolism, is upregulated by proline but downregulated by dehydration in Arabidopsis.

Proline is one of the most common compatible osmolytes in water-stressed plants. The accumulation of proline in dehydrated plants is caused both by the activation of proline biosynthesis and by the inactivation of proline degradation; a decrease in the level of accumulated proline in rehydrated plants is caused both by the inhibition of proline biosynthesis and by the activation of proline degradation. The proline biosynthetic pathway has been well characterized, but the degradation of proline is poorly understood. Sequence analysis of an Arabidopsis cDNA clone, ERD5 (for early responsive to dehydration stress), isolated from plants dehydrated for 1 hr, revealed that it encodes a protein with identity to products of the yeast PUT1 (for proline utilization) gene (23.6% over 364 amino acids) and the Drosophila sluggish-A gene (34.5% over 255 amino acids). Their gene products are precursors of proline oxidases (dehydrogenase) (EC 1.5.99.8), which are the first enzymes involved in the conversion of proline to glutamic acid. Proline oxidase is localized in mitochondria. RNA gel blot analysis demonstrated that transcripts of the ERD5 gene were undetectable when plants had been dehydrated for 10 hr, but large amounts of the transcript accumulated when plants subsequently were rehydrated. Elevated levels of the transcript were also found in plants that had been incubated in a medium that contained proline. Immunologically, we showed that the product of ERD5 is localized in the mitochondrial fraction and accumulates in response to proline in cultured cells. Fusion genes for ERD5 and PUT1 complemented a put1 mutant of yeast, allowing put1 to grow with proline as the source of nitrogen. These results suggest that ERD5 encodes a precursor of proline dehydrogenase (oxidase), which is regulated at the level of mRNA accumulation in both dehydrated and rehydrated plants.

Role of CodY in regulation of the Bacillus subtilis hut operon

Bacillus subtilis mutants deficient in amino acid repression of the histidine utilization (hut) operon were isolated by transposon mutagenesis. Genetic characterization of these mutants indicated that they most likely contained transposon insertions within the codVWXY operon. The codY gene is required for nutritional regulation of the dipeptide permease (dpp) operon. An examination of hut expression in a delta codY mutant demonstrated that amino acid repression exerted at the hutOA operator, which lies immediately downstream of the hut promoter, was defective in a delta codY mutant. The codY gene product was not required for amino acid regulation of either hut induction or the expression of proline oxidase, the first enzyme in proline degradation. This indicates that more than one mechanism of amino acid repression is present in B. subtilis. An examination of dpp and hut expression in cells during exponential growth in various media revealed that the level of CodY-dependent regulation appeared to be related to the growth rate of the culture.

CHANGES IN LEAF WATER POTENTIAL, OSMOTIC ADJUSTMENT, AND PROLINE METABOLISM IN MULBERRY DURING WATER STRESS

The influence of water stress on amino acid content and proline metabolism was studied in 3-month-old mulberry plants at four levels of water stress. Leaf water potential and osmotic potential were significantly decreased in all stress treatments. Though leaf area and relative water content decreased, significantly, a marked decrease was observed only in severe stress treatment. Accumulation of total amino acids and proline was observed both in roots and leaves in stress treatments, with amino acid accumulation greater in leaves than in roots. In contrast, proline accumulation was greater in roots than in leaves. Activities of proline dehydrogenase and proline oxidase were inhibited under stress conditions. Inhibition of proline oxidase was greater in roots than in leaves.

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

ABSTRACTNostoc muscorum required an active proline oxidase in order to assimilate exogenous proline as a source of fixed nitrogen. A mutant strain (Acr) 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 Acr phenotype or from salinity‐inducible uptake of exogenous proline, conferred enhanced salinity tolerance in this cyanobacterium.

The roles of PUT3, URE2, and GLN3 regulatory proteins in the proline utilization pathway ofSaccharomyces cerevisiae

The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as allantoin, urea, γ-aminobutyrate, or proline when preferred nitrogen sources such as asparagine, glutamine, or ammonium ions are unavailable in the environment. To use proline as the sole nitrogen source, cells must activate the expression of the proline transporters and the genes that encode the catabolic enzymes proline oxidase (PUT1) and Δ1-pyrroline-5-carboxylate dehydrogenase (PUT2). Transcriptional activation of the PUT genes requires the PUT3 regulatory protein, proline, and relief from nitrogen repression. PUT3 is a 979 amino acid protein that binds a short DNA sequence in the promoters of PUT1 and PUT2, independent of the presence of proline. The functional domains of PUT3 have been studied by biochemical and molecular tests and analysis of activator-constitutive and activator-defective mutant proteins. Mutations in the URE2 gene relieve nitrogen repression, permitting inducer-independent transcription of the PUT genes in the presence of repressing nitrogen sources. The GLN3 protein that activates the expression of many genes in alternative nitrogen source pathways is not required for the expression of the PUT genes under inducing, derepressing conditions (proline) or noninducing, repressing conditions (ammonia). Although it has been speculated that the URE2 protein antagonizes the action of GLN3 in the regulation of many nitrogen assimilatory pathways, URE2 appears to act independently of GLN3 in the proline-utilization pathway. Key words: Saccharomyces cerevisiae, proline utilization, nitrogen repression.