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.     

Expression of delta1-pyrroline-5-carboxylate synthetase gene during drought in rice (Oryza sativa L.)

The 45-days-old seedlings of drought resistant (N-22, CR143-2-2) and susceptible rice (Oryza sativa L.) genotypes (Panidhan, Pusa-169) were subjected to osmotic stress in PEG-6000 solution of -10 and -16 bar and the relative water content (RWC), proline content, and pyrroline-5-carboxylate synthetase (P5CS) activity and its P5CS expression were studied. A gradual decrease in RWC was observed in tolerant genotypes, whereas the decrease was drastic in susceptible ones. Proline content and P5CS activity increased both in susceptible and tolerant genotypes; the increase was higher in tolerant genotypes. Higher proline levels in tolerant genotypes were due to increased P5CS activity. The EcoRI, BamHI and XbaI restricted DNA of N-22 and Panidhan genotypes were hybridized with Arabidopsis P5CS sequence and a single band (approx 2.4 kb) was observed, however, P5CS expression was more in N-22 as compared to Panidhan.     

Delta-1-pyrroline-5-carboxylate and delta-1-pyrroline-3-hydroxy-5-carboxylate: chromatography on the amino acid analyzer.

The chromatographhy of Δ¹-pyrroline-5-carboxylate and Δ¹-pyrroline-3-hydroxy-5-carboxylate under routine amino acid analyzer conditions is described. The former compound is unstable under these conditions and is recovered intact from the analyzer column in only 30–40% yield, together with a single major peak which has lost much or all of the initial substrate activity with a specific reductase, but still reacts with ninhydrin and, to a lesser extent, with o-aminobenzaldehyde. Δ¹-Pyrroline-3-hydroxy-5-carboxylate is much more stable and can be conveniently measured by this procedure, even in the presence of hydroxyproline, with which it co-elutes in the present system.     

The role of [Delta]1-pyrroline-5-carboxylate dehydrogenase in proline degradation

In response to stress, plants accumulate Pro, requiring degradation after release from adverse conditions. Delta1-Pyrroline-5-carboxylate dehydrogenase (P5CDH), the second enzyme for Pro degradation, is encoded by a single gene expressed ubiquitously. To study the physiological function of P5CDH, T-DNA insertion mutants in AtP5CDH were isolated and characterized. Although Pro degradation was undetectable in p5cdh mutants, neither increased Pro levels nor an altered growth phenotype were observed under normal conditions. Thus AtP5CDH is essential for Pro degradation but not required for vegetative plant growth. External Pro application caused programmed cell death, with callose deposition, reactive oxygen species production, and DNA laddering, involving a salicylic acid signal transduction pathway. p5cdh mutants were hypersensitive toward Pro and other molecules producing P5C, such as Arg and Orn. Pro levels were the same in the wild type and mutants, but P5C was detectable only in p5cdh mutants, indicating that P5C accumulation may be the cause for Pro hypersensitivity. Accordingly, overexpression of AtP5CDH resulted in decreased sensitivity to externally supplied Pro. Thus, Pro and P5C/Glu semialdehyde may serve as a link between stress responses and cell death.     

Metabolic regulation of the gene encoding glutamine-dependent asparagine synthetase in Arabidopsis thaliana.

Here, we characterize a cDNA encoding a glutamine-dependent asparagine synthetase (ASN1) from Arabidopsis thaliana and assess the effects of metabolic regulation on ASN1 mRNA levels. Sequence analysis shows that the predicted ASN1 peptide contains a purF-type glutamine-binding domain. Southern blot experiments and cDNA clone analysis suggest that ASN1 is the only gene encoding glutamine-dependent asparagine synthetase in A. thaliana. The ASN1 gene is expressed predominantly in shoot tissues, where light has a negative effect on its mRNA accumulation. This negative effect of light on ASN1 mRNA levels was shown to be mediated, at least in part, via the photoreceptor phytochrome. We also investigated whether light-induced changes in nitrogen to carbon ratios might exert a metabolic regulation of the ASN1 mRNA accumulation. These experiments demonstrated that the accumulation of ASN1 mRNA in dark-grown plants is strongly repressed by the presence of exogenous sucrose. Moreover, this sucrose repression of ASN1 expression can be partially rescued by supplementation with exogenous amino acids such as asparagine, glutamine, and glutamate. These findings suggest that the expression of the ASN1 gene is under the metabolic control of the nitrogen to carbon ratio in cells. This is consistent with the fact that asparagine, synthesized by the ASN1 gene product, is a favored compound for nitrogen storage and nitrogen transport in dark-grown plants. We have put forth a working model suggesting that when nitrogen to carbon ratios are high, the gene product of ASN1 functions to re-direct the flow of nitrogen into asparagine, which acts as a shunt for storage and/or long-distance transport of nitrogen.     

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.     

Li+-regulated 1-aminocyclopropane-1-carboxylate synthase gene expression in Arabidopsis thaliana

In Arabidopsis thaliana, 1-aminocyclopropane-1-carboxylate synthase (ACS) is encoded by a multigene family consisting of at least five members whose expression is induced by hormones, developmental signals, and protein synthesis inhibition. Li⁺, known to interfere with the phosphoinositide (PI) second messenger system by inhibiting the activity of inositol-phosphate phosphatases, is one of the strongest inducers of ACC synthase activity in plants. Treatment of etiolated Arabidopsis seedlings with LiCl results in a rapid induction of the ACS5 gene. Also, LiCl represses the cycloheximide (CHX)-induced accumulation of the ACS2 mRNA. The effects of Li⁺ on the expression of ACS5 and ACS2 are specific, dose-dependent, and can be reversed by Ca²⁺ and mimicked by the protein kinase inhibitor K-252a. The results suggest that the regulation of some ACS genes by various inducers may involve protein kinase activity, which in turn may be controlled through an inositol 1,4,5-triphosphate (IP₃)-mediated Ca²⁺ mobilization. Since plants contain no Li⁺, the cation appears to unmask pre-existing biochemical capacity that may be utilized by various unknown transducers during plant growth and development.     

Oscillation and regulation of proline content by P5CS and ProDH gene expressions in the light/dark cycles in Arabidopsis thaliana L

The fluctuation of proline content, and protein and mRNA levels of delta1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH), both of which are involved in proline biosynthesis and degradation, in the shoots of Arabidopsis grown in light/dark cycles were demonstrated under salt-stressed and unstressed conditions. Proline content, as well as proteins and mRNAs of these enzymes, clearly oscillated in the light/dark cycles under the stressed and unstressed conditions. A reciprocal relationship between P5CS and ProDH was observed. Protein levels of P5CS and ProDH were well synchronized with their mRNA levels, although the fluctuation of protein levels was not as significant as that of their mRNA levels. Both mRNA and protein levels of the two enzymes as well as the proline content did not oscillate under the continuous light or the dark conditions. Thus, P5CS and ProDH gene expressions seemed to be involved in light irradiation. Moreover, relative water content (RWC) in the plants oscillated in the light/dark cycles. The fluctuations of proline content in shoot reversely responded to that of RWC. It is suggested that the expression of two genes responds sensitively to a subtle change of cellular water status, and accumulated proline keeps the osmotic balance between cells and the outer environment.     

Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana

The plant, Arabidopsis thaliana, contains two S-adenosylmethionine synthetase-encoding genes (sam). Here, we analyze the structure and expression of the sam-2 gene and compare it with the previously described sam-1 gene. Northern-blot analysis using gene-specific probes shows that both sam-1 and sam-2 are highly expressed in stem, root, and callus tissue. This similar expression pattern might be mediated by the presence of three highly conserved sequences in the 5' region of both sam genes. Using a chimeric beta-glucuronidase (GUS)-encoding gene, we show that in transgenic tobacco plants, 748 bp of 5' sam-1 sequences generate high GUS activity in the same type of tissues as previously observed in transgenic A. thaliana plants. A deletion analysis of these 5' sam-1 sequences indicates that 224 bp of 5' sam-1 sequences can still induce higher expression of the gene in stem and root relative to leaf. However, the level of expression is reduced when compared to the expression level obtained with the full-length promoter.     

Characteristics of delta 1-pyrroline-5-carboxylate reductase from Drosophila melanogaster.

1. Biochemical properties of delta 1-pyrroline-5-carboxylate reductase from d. melanogaster have been investigated. 2. The enzyme is stable below 4 degrees C. 3. the pH optimum of the enzyme is 5.7. It is rapidly inactivated below pH 5.4. 4. The Km values for NADPH and delta 1-pyrroline-5-carboxylate are 1.6 x 10-5 and 2.5 x 10-6 M, respectively. 5. the estimated molecular weight of the enzyme is 225,000. 6. the enzyme is weakly inhibited by L-proline (Ki = 0.12 M).     

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.     

Escherichia coli delta 1-pyrroline-5-carboxylate reductase: gene sequence, protein overproduction and purification.

The sequence of the Escherichia coli proC gene which encodes for delta 1-pyrroline-5-carboxylate (PCA) reductase was determined. Overproduction of the proC gene product via an expression plasmid carrying the bacteriophage lambda PL promoter allowed the purification to homogeneity of PCA reductase by affinity adsorption chromatography. NH2 and COOH-terminal analysis and amino acid composition of the purified proC protein is consistent with the gene sequence reported. The molecular weight of the proC monomer is 28,112.     

Possible involvement of a L-delta 1-pyrroline-5-carboxylate (P5C) reductase in the synthesis of proline in Desulfovibrio desulfuricans Norway

A L-delta 1-pyrroline-5-carboxylate reductase activity has been detected in crude extracts of Desulfovibrio desulfuricans Norway. This P5C reductase activity is also found when a 2.5 kb D. desulfuricans DNA fragment is introduced into an Escherichia coli proC mutant. Although it restores growth of the proC mutant, the ProDd enzyme might be detrimental to the E. coli host since the plasmid carrying the cognate proDd gene is segregated at high rate by the cells but is stabilized by small deletions which lead to a loss of the P5C reductase activity.     

Removal of feedback inhibition of delta(1)-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress

The Delta(1)-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned) is the rate-limiting enzyme in proline (Pro) biosynthesis in plants and is subject to feedback inhibition by Pro. It has been suggested that the feedback regulation of P5CS is lost in plants under stress conditions. We compared Pro levels in transgenic tobacco (Nicotiana tabacum) plants expressing a wild-type form of Vigna aconitifolia P5CS and a mutated form of the enzyme (P5CSF129A) whose feedback inhibition by Pro was removed by site-directed mutagenesis. Transgenic plants expressing P5CSF129A accumulated about 2-fold more Pro than the plants expressing V. aconitifolia wild-type P5CS. This difference was further increased in plants treated with 200 mM NaCl. These results demonstrated that the feedback regulation of P5CS plays a role in controlling the level of Pro in plants under both normal and stress conditions. The elevated Pro also reduced free radical levels in response to osmotic stress, as measured by malondialdehyde production, and significantly improved the ability of the transgenic seedlings to grow in medium containing up to 200 mM NaCl. These findings shed new light on the regulation of Pro biosynthesis in plants and the role of Pro in reducing oxidative stress induced by osmotic stress, in addition to its accepted role as an osmolyte.