Crystal structure of Thermus thermophilus Delta1-pyrroline-5-carboxylate dehydrogenase

Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDh) plays an important role in the metabolic pathway from proline to glutamate. It irreversibly catalyzes the oxidation of glutamate-gamma-semialdehyde, the product of the non-enzymatic hydrolysis of Delta(1)-pyrroline-5-carboxylate, into glutamate with the reduction of NAD(+) into NADH. We have confirmed the P5CDh activity of the Thermus thermophilus protein TT0033 (TtP5CDh), and determined the crystal structure of the enzyme in the ligand-free form at 1.4 A resolution. To investigate the structural basis of TtP5CDh function, the TtP5CDh structures with NAD(+), with NADH, and with its product glutamate were determined at 1.8 A, 1.9 A, and 1.4 A resolution, respectively. The solved structures suggest an overall view of the P5CDh catalytic mechanism and provide insights into the P5CDh deficiencies in the case of the human type II hyperprolinemia.     

Enzymes metabolizing delta1-pyrroline-5-carboxylate in rat tissues.

The direction and capacity for the metabolism of delta1-pyrroline-5-carboxylate in a number of rat tissues ere investigated by measuring the activities of delta1-pyrroline-5-carboxylate reductase, delta1-pyrroline-5-carboxylate dehydrogenase and proline oxidase. Each of these enzymes catalyzed unidirectional reactions in which delta1-pyrroline-5-carboxylate was either the substrate or product. Delta1-Pyrroline-5-carboxylate reductase activities that were much higher than any previously reported were obtained by avoiding its inactivation in the cold. delta1-Pyrroline-5-carboxylate dehydrogenase, previously said to act on both D- and L-isomers of delta1-pyrroline-5-carboxylate, acted only on the L-isomer. Proline oxidase could not be measured in two adult tissues, in which an inhibitor appeared after birth. The activity of delta1-pyrroline-5-carboxylate reductase significantly paralleled that of ornithine aminotransferase in 23 tissues, showing a widespread potential for proline synthesis from ornithine. An independently distributed potential in fewer tissues for proline degradation to alpha-oxoglutarate was shown by the significantly similar tissue distributions of proline oxidase. Delta1-pyrroline-5-carboxylate dehydrogenase and glutamate dehydrogenase. Reverse metabolism of glutamate or proline to ornithine would be atypical in rat tissues with these distributions of unidirectional enzyme reactions.     

Assay and subcellular localization of pyrroline-5-carboxylate dehydrogenase in rat liver

Delta1-pyrroline-5-carboxylate dehydrogenase (P5CDh) catalyzes the conversion of Delta1-pyrroline-5-carboxylate to glutamate in a reaction requiring NADP+ as a cofactor. Delta1-pyrroline-5-carboxylate is formed in liver from proline by proline oxidase (EC number not assigned) or from ornithine via ornithine aminotransferase. A spectrophotometric assay for P5CDh was shown to be valid if rotenone was included in the assay to prevent reoxidation of NADH. Using this new assay, liver was fractionated using differential centrifugation and the distribution of P5CDh was compared to that of appropriate marker enzymes. P5CDh is enriched only in the mitochondrial fractions, as are the mitochondrial enzymes, succinate cytochrome c reductase, proline oxidase, glutaminase, and ornithine aminotransferase. Thus, it can be concluded that P5CDh occurs only in mitochondria, not in both mitochondria and cytoplasm, as had previously been reported.     

Regulation of proline catabolism in Pseudomonas aeruginosa PAO

Mutants of Pseudomonas aeruginosa deficient in the utilization of l-proline as the only carbon and nitrogen source have been found to be defective either in proline dehydrogenase activity or in both proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities of the bifunctional proline degradative enzyme. The latter type of mutants was unable to utilize l-ornithine, indicating that a single ¹-pyrroline-5-carboxylate dehydrogenase activity is involved in the degradation of ornithine and proline. Proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities were strongly and coordinately induced by proline. It was excluded that ¹-pyrroline-5-carboxylate acted as an inducer of the bifunctional enzyme and it was shown that the low level induction observed during growth on ornithine was due to the intracellular formation of proline. The formation of the proline degradative enzyme was shown to be subject to catabolite repression by citrate and nitrogen control.Abbreviations EMS Ethylmethane sulfonate - NG N-methyl-N-nitro-N-nitrosoguanidine - P Minimal medium P - Pro-DH Proline dehydro-genase - P5C ¹-Pyrroline-5-carboxylate - P5C-DH ¹-Pyrroline-5-carboxylate dehydrogenase     

Exogenous ornithine is an effective precursor and the δ-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves

The role of the δ-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Δ¹-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS, GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Δ¹-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves.     

Purification and characterization of Delta(1)-pyrroline-5-carboxylate reductase isoenzymes, indicating differential distribution in spinach (Spinacia oleracea L.) leaves

Delta(1)-Pyrroline-5-carboxylate reductase (P5CR) (EC 1.5.1.2. L-proline: NAD(P)-5-oxidoreductase), the second enzyme in the proline biosynthetic pathway, was purified from spinach (Spinacia oleracea L.) leaves. Following ammonium sulfate fractionation, purification was performed by several chromatographic methods: Blue Cellulofine, DEAE-TOYOPEARL, Sephacryl S-300 HR, and POROS QE/M. Two isoenzymes resolved by anion exchange chromatography were designated P5CR-1 and P5CR-2. Only P5CR-2 was purified from the intact chloroplasts, indicating differential distribution of the isoenzymes. P5CR isoenzymes, P5CR-1 and P5CR-2, are a homopolymer with an apparent molecular mass of 310 kDa, consisting of 10 to 12 subunits of about 28.5 kDa. P5CR-1 and P5CR-2 showed K(m) values of 9 and 19 microM for NADPH and values of 0.122 and 0.162 mM for Delta(1)-pyrroline-5-carboxylate (P5C), respectively. We decided partial amino acid sequences of P5CR-1 which showed the 70 to 80% homology to the deduced amino acid sequences of several plant P5CR cDNAs. Both isoenzymes had much lower affinity for NADH than for NADPH and were inhibited by free ATP and Mg(2+) ion. The inhibition was partially mitigated when ATP and Mg(2+) were added simultaneously to the reaction mixture. Cations at high concentration were inhibitory to P5CR activity. Interestingly, P5CR-2 was more stable to heat treatment at 40 degrees C than P5CR-1.     

Proline: an essential intermediate in arginine degradation in Saccharomyces cerevisiae.

Results of studies on proline-nonutilizing (Put-) mutants of the yeast Saccharomyces cerevisiae indicate that proline is an essential intermediate in the degradation of arginine. Put- mutants excreted proline when grown on arginine or ornithine as the sole nitrogen source. Yeast cells contained a single enzyme, delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase, which is essential for the complete degradation of both proline and arginine. The sole inducer of this enzyme was found to be proline. P5C dehydrogenase converted P5C to glutamate, but only when the P5C was derived directly from proline. When the P5C was derived from ornithine, it was first converted to proline by the enzyme P5C reductase. Proline was then converted back to P5C and finally to glutamate by the Put enzymes proline oxidase and P5C dehydrogenase.     

Glutamine synthetase and glutamate dehydrogenase contribute differentially to proline accumulation in leaves of wheat (Triticum aestivum) seedlings exposed to different salinity

To investigate the roles of ammonium-assimilating enzymes in proline synthesis under salinity stress, the activities of glutamine synthetase (GS; EC 6.3.1.2) and NADH-dependent glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) were determined in leaves of wheat (Triticum aestivum) seedlings exposed to salt stress at 150 and 300 mM NaCl for 5d. At the lower salinity, only GS activity increased markedly. At 300 mM NaCl, however, NADH-GDH activity increased while GS activity decreased. A significant accumulation of proline was found only at high-salinity exposure while glutamate, a proline precursor, increased dramatically under both low and high salinity. These data suggests that GS-catalysis might be the main glutamate synthesis pathway under low salinity. At 300 mM NaCl, glutamate seems to be preferentially produced through the process catalyzed by NADH-GDH. The increase of ammonium in salinity-stressed wheat seedlings might have resulted from increased photorespiration, which is responsible for the higher NADH-GDH activity. The activity of Delta(1)-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) was significantly enhanced at 300 mM NaCl but remained unchanged at 150 mM. Delta(1)-Pyrroline-5-carboxylate synthetase (P5CS) activity did not show a specific response, indicating that P5CR might be the limiting step in proline synthesis from glutamate at high salinity.     

Hg and Cd Induced Changes in Proline Content and Activities of Proline Biosynthesizing Enzymes in Phaseolus Aureus and Triticum Aestivum

The effect of mercury and cadmium, in the form of HgCl₂ and CdCl₂ respectively, on proline accumulation and two key proline biosynthesizing enzymes, ¹-pyrroline-5-carboxylate synthetase (P5CS) and ¹-pyrroline-5-carboxylate reductase (P5CR), was investigated in Phaseolus aureus Roxb. and Triticum aestivum L. The 5-d-old seedlings were exposed to 0.05, 0.1, 0.2 or 0.4 mM concentrations of the metals in Hoagland solution for 12 and 36 h. T. aestivum exhibited considerably greater accumulation of proline than P. aureus in response to the metal treatment. Among the two metals, Hg induced greater accumulation of proline than Cd. The activity of P5CS increased significantly in response to the metal treatment, particularly in T. aestivum in which the activity of the enzyme in the control was much higher than that was in P. aureus. The activity of P5CR on the other hand mostly decreased in response to the metal treatment. The study indicated a strong dependence of the metal induced proline accumulation on the constitutive P5CS content of the plants.     

A radioisotopic assay for delta 1-pyrroline-5-carboxylate synthase activity

A radioisotopic assay is described for measuring the activity of delta 1-pyrroline-5-carboxylate synthase, the enzyme that catalyzes the formation of delta 1-pyrroline-5-carboxylic acid from glutamic acid. Pyrroline-5-carboxylic acid is a common intermediate in the pathways through which glutamic acid, proline, and ornithine are interconverted. To determine pyrroline-5-carboxylate synthase activity, cell homogenates are incubated with [14C]-glutamic acid, the products of the reaction are converted quantitatively to proline by sodium borohydride, and proline is isolated by cation-exchange column chromatography. Cofactor requirements have been defined, and the activity of pyrroline-5-carboxylate synthase in several different cultured fibroblast lines is reported.     

Regional Distribution in Rat Brain of 1-Pyrroline-5-Carboxylate Dehydrogenase and Its Localization to Specific Glial Cells

A possible alternative route for production of a small glutamate pool in brain is from proline or ornithine to 1-pyrroline-5-carboxylate (P5C) and thence to glutamate. The conversion from ornithine to P5C is catalyzed by ornithine delta-aminotransferase (OrnT) whereas that from proline is catalyzed by proline oxidase (PrO). The conversion of P5C to glutamate is catalyzed by 1-pyrroline-5-carboxylate dehydrogenase (PDH). Biochemical assays of PDH and PrO in various rat brain regions indicate no positive correlation between the two enzymes nor between either activity and high-affinity glutamate uptake or the regional distribution of OrnT. We have localized PDH and PrO histochemically by modifications of the Van Gelder [J. Neurochem. 12, 231-237, (1965)] method for gamma-aminobutyric acid (GABA) transaminase. The enzymes were found only in certain types of glial cells; the best stained were the Bergmann glial cells of the cerebellum but, for PDH, there was also good staining of astrocytes in the dentate area of the hippocampus. Since both these areas are believed to have heavy glutamate innervation and numerous GABA interneurons, these findings may reflect an alternative route of glutamate production in glial cells near some glutamate and/or GABA tracts but they do not support this as a possible route for glutamate formation in most brain regions. The findings do, however, provide further evidence for chemical specialization of glial cells.     

Aldehyde oxidation in human placenta. Purification and properties of 1-pyrroline-5-carboxylate dehydrogenase.

The human placenta contains a considerable amount of 1-pyrroline-5-carboxylate dehydrogenase (23 +/- 6 micrograms/g; n = 12), about 25% of the concentration present in liver. The enzyme is the only form in placenta that oxidizes short- and medium-chain aldehydes, which facilitates its purification from this organ. It can be purified to homogeneity by successive chromatographies on DEAE-cellulose, 5'-AMP-Sepharose and Sephacryl S-300. From 500 g of tissue, about 2.1 units of enzyme can be obtained with a 12% yield. Placental 1-pyrroline-5-carboxylate dehydrogenase is a dimer of Mr-63,000 subunits. It exhibits a pI of 6.80-6.65, and is specific for 1-pyrroline-5-carboxylate, the cyclic form of glutamate gamma-semialdehyde (Km = 0.17 mM, kcat. = 870 min-1), although it also oxidizes short-chain aliphatic aldehydes such as propionaldehyde (Km = 24 mM, kcat. = 500 min-1). These properties are very close to those of the liver enzyme, indicating a strong similarity between the enzyme forms from both organs. The enzyme is highly sensitive to temperature, showing 50% inhibition after incubation for 0.8 min at 45 degrees C or after 23 min at 25 degrees C. It is irreversibly inhibited by disulfiram, and a molar ratio inhibitor: enzyme of 60:1 produced 50% inhibition after incubation for 10 min. A subcellular-distribution study indicates that the enzyme is located in two compartments: the mitochondria, with 60% of the total activity, and the cytosol, with 40% activity. The physiological role of the enzyme in placental amino acid metabolism is discussed.     

Urea cycle of Fasciola gigantica: purification and characterization of arginase

The ornithine-urea cycle has been investigated in Fasciola gigantica. Agrinase had very high activity compared to the other enzymes. Carbamoyl phosphate synthetase and ornithine carbamoyltransferase had very low activity. A moderate enzymatic activity was recorded for argininosuccinate synthetase and argininosuccinate lyase. The low levels of F. gigantica urea cycle enzymes except to the arginase suggest the urea cycle is operative but its role is of a minor important. The high level of arginase activity may benefit for the hydrolysis of the exogenous arginine to ornithine and urea. Two arginases Arg I and Arg II were separated by DEAE-Sepharose column. Further purification was restricted to Arg II with highest activity. The molecular weight of Arg II, as determined by gel filtration and SDS-PAGE, was 92,000. The enzyme was capable to hydrolyze l-arginine and to less extent l-canavanine at arginase:canavanase ratio (>10). The enzyme exhibited a maximal activity at pH 9.5 and Km of 6 mM. The optimum temperature of F. gigantica Arg II was 40 degrees C and the enzyme was stable up to 30 degrees C and retained 80% of its activity after incubation at 40 degrees C for 15 min and lost all of its activity at 50 degrees C. The order of effectiveness of amino acids as inhibitors of enzyme was found to be lysine>isoleucine>ornithine>valine>leucine>proline with 67%, 43%, 31%, 25%, 23% and 15% inhibition, respectively. The enzyme was activated with Mn2+, where the other metals Fe2+, Ca2+, Hg2+, Ni2+, Co2+ and Mg2+ had inhibitory effects.     

Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.     

Subcellular compartmentation in control of converging pathways for proline and arginine metabolism in Saccharomyces cerevisiae.

Enzymes of proline biosynthesis and proline degradation which act on the same compound, delta 1-pyrroline-5-carboxylate, are physically separated in yeast cells. The enzyme responsible for the final step in proline biosynthesis, pyrroline-5-carboxylate reductase, converts pyrroline-5-carboxylate to proline and is located in the cytoplasm. The last enzyme in the proline degradative pathway, pyrroline-5-carboxylate dehydrogenase, converts pyrroline-5-carboxylate to glutamate and is found in the particulate fraction of the cell, presumably in the mitochondrion. By subcellular compartmentation, yeast cells avoid futile cycling between proline and pyrroline-5-carboxylate.     

The proline biosynthesis in living organisms

Summary In this article we review recent work on the physiology of proline and ¹-pyrroline-5-carboxylate (P5C) in living organisms and consider recent progress in our understanding of the role of P5C synthetase in collagen metabolism and the regulation of urea cycle in vertebrates. Much of this recent progress has been made possible by advances in our knowledge of the enzymes and genes involved in proline biosynthesis in man. The availability of well characterized P5C synthetase deficiency in man has been an impetus for the cloning of the cDNA encoding for this enzyme from man and facilitated the establishment of the phenotype-genotype relationships in P5C synthetase deficiency in higher vertebrates.Abbreviations GK -glutamyl kinase - GPR -glutamyl phosphate reductase - P5CR ¹-pyrroline-5-carboxylate reductase - GSA glutamic--semialdehyde - P5C ¹-pyrroline-5-carboxylate - P₁ Inorganic phosphate - AMP, ADP, ATP Adenosine 5-mono-, di-, triphosphate - NAD⁺, NADH nicotinamide adenine dinucleotide, and its reduced form - NADP⁺, NADPH nicotinamide adenine dinucleotide phosphate, and its reduced form; DEAF: diethylaminoethyle - OAT ornithine amino transferase; CHO: Chinese hamster ovary - IGF-1 insulin-like growth factor-1 - P5CDH pyrroline 5carboxylate dehydrogenase - IMP inosine 5-monophosphate     

Fungal metabolism of biphenyl.

gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.     

Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult

Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively.