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.
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.
Proline metabolism in mammals involves two other amino acids, glutamate and ornithine, and five enzymatic activities, Δ1-pyrroline-5-carboxylate (P5C) reductase (P5CR), proline oxidase, P5C dehydrogenase, P5C synthase and ornithine-δ-aminotransferase (OAT). With the exception of OAT, which catalyzes a reversible reaction, the other 4 enzymes are unidirectional, suggesting that proline metabolism is purpose-driven, tightly regulated, and compartmentalized. In addition, this tri-amino-acid system also links with three other pivotal metabolic systems, namely the TCA cycle, urea cycle, and pentose phosphate pathway. Abnormalities in proline metabolism are relevant in several diseases: six monogenic inborn errors involving metabolism and/or transport of proline and its immediate metabolites have been described. Recent advances in the Human Genome Project, in silico database mining techniques, and research in dissecting the molecular basis of proline metabolism prompted us to utilize functional genomic approaches to analyze human genes which encode proline metabolic enzymes in the context of gene structure, regulation of gene expression, mRNA variants, protein isoforms, and single nucleotide polymorphisms.
Apoptosis; FASTSNP; Functional genomics; OAT; OH-POX; OMIM; P53; Δ1-pyrroline-5-carboxylate (P5C); P5CDH; P5CR/PYCR; P5CS/PYCS; POX/PRODH; L-Proline; Promoter analysis; SNP
The initial step in the degradation pathways of proline and hydroxyproline is catalyzed by proline oxidase and hydroxyproline oxidase, yielding delta 1-pyrroline-5-carboxylate and delta 1-pyrroline-3-hydroxy-5-carboxylate, respectively. The second step is the oxidation of delta 1-pyrroline-5-carboxylate to glutamate and delta 1-pyrroline-3-hydroxy-5-carboxylate to gamma-hydroxy-glutamate. To determine if this second step in the degradation of proline and hydroxyproline is catalyzed by a common or by separate enzyme(s), we developed a radioisotopic assay for delta 1-pyrroline-3-hydroxy-5-carboxylate dehydrogenase activity. We then compared delta1-pyrroline-3-hydroxy-5-carboxylate dehydrogenase activity with that of delta 1-pyrroline-5-carboxylate dehydrogenase in fibroblasts and leukocytes from type II hyperprolinemia patients, heterozygotes, and controls. We found that cells from type II hyperprolinemia patients were deficient in both dehydrogenase activities. Furthermore, these activities were highly correlated over the range found in the normals, heterozygotes, and patients. We conclude from these data that a common delta 1-pyrroline-5-carboxylate dehydrogenase catalyzes the oxidation of both delta 1-pyrroline-5-carboxylate and delta 1-pyrroline-3-hydroxy-5-carboxylate, and that this activity is deficient in type II hyperprolinemia.
L-Proline is an amino acid that plays an important role in proteins uniquely contributing to protein folding, structure, and stability, and this amino acid serves as a sequence-recognition motif. Proline biosynthesis can occur via two pathways, one from glutamate and the other from arginine. In both pathways, the last step of biosynthesis, the conversion of Δ1-pyrroline-5-carboxylate (P5C) to L-proline, is catalyzed by Δ1-pyrroline-5-carboxylate reductase (P5CR) using NAD(P)H as a cofactor. We have determined the first crystal structure of P5CR from two human pathogens, Neisseria meningitides and Streptococcus pyogenes, at 2.0Å and 2.15Å resolution, respectively. The catalytic unit of P5CR is a dimer composed of two domains, but the biological unit seems to be species-specific. The N-terminal domain of P5CR is an α/β/α sandwich, a Rossmann fold. The C-terminal dimerization domain is rich in α-helices and shows domain swapping. Comparison of the native structure of P5CR to structures complexed with L-proline and NADP+ in two quite different primary sequence backgrounds provides unique information about key functional features: the active site and the catalytic mechanism. The inhibitory L-proline has been observed in the crystal structure.
structural genomics; MAD phasing; SAD phasing; proline biosynthesis; P5C reductase
Proline-requiring mutants of Saccharomyces cerevisiae were isolated. Each mutation is recessive and is inherited as expected for a single nuclear gene. Three complementation groups cold be defined which are believed to correspond to mutations in the three genes (pro1, pro2, and pro3) coding for the three enzymes of the pathway. Mutants defective in the pro1 and pro2 genes can be satisfied by arginine or ornithine as well as proline. This suggests that the blocks are in steps leading to glutamate semialdehyde, either in glutamyl kinase or glutamyl phosphate reductase. A pro3 mutant has been shown by enzyme assay to be deficient in delta 1-pyrroline-5-carboxylate reductase which converts pyrroline-5-carboxylate to proline. A unique feature of yeast proline auxotrophs is their failure to grown on the rich medium, yeast extract-peptone-glucose. This failure is not understood at present, although it accounts for the absence of proline auxotrophs in previous screening for amino acid auxotrophy.
The PRO3 gene of Saccharomyces cerevisiae encodes the 286-amino-acid protein delta 1-pyrroline-5-carboxylate reductase [L-proline:NAD(P+) 5-oxidoreductase; EC 184.108.40.206], which catalyzes the final step in proline biosynthesis. The protein has substantial similarity to the pyrroline carboxylate reductases of diverse bacterial species, soybean, and humans. Using RNA hybridization and measurements of enzyme activity, we have determined that the expression of the PRO3 gene appears to be constitutive. It is not repressed by the pathway end product (proline), induced by the initial substrate (glutamate), or regulated by the general control system. Its expression is not detectably altered when cells are grown in a wide range of nitrogen sources or when glycerol and ethanol replace glucose as the carbon source. The possibility that this enzyme has other functions in addition to proline biosynthesis is discussed.
The last step in proline biosynthesis in Escherichia coli K-12, Salmonella typhimurium LT7, and a number of other enterobacterial isolates is regulated so that no proline is excreted, even if excess Δ1-pyrroline-5-carboxylate, the immediate precursor of proline, is added to a culture. In proline auxotrophs blocked at an early step in proline biosynthesis (proA or proB), reversion to prototrophy is often due to a mutation in the arginine pathway which diverts N-acetyl glutamate γ-semialdehyde to proline synthesis, thus bypassing the proA or proB block. In such double mutants (proAB, argD), the last step in proline synthesis appears to be unregulated, since proline is excreted. Feedback inhibition and repression of the arginine pathway overcomes indirect suppression (restoring the Pro− phenotype), but proline regulation is not restored; double mutants still excrete proline when fed Δ1-pyrroline-5-carboxylate exogeneously. A new class of proline analogue-resistant mutant, due to mutation at argD, is also described.
Mammalian Δ1-pyrroline-5-carboxylate synthase (P5CS) is a bifunctional ATP- and NAD(P)H-dependent mitochondrial enzyme that catalyzes the coupled phosphorylation and reduction-conversion of L-glutamate to P5C, a pivotal step in the biosynthesis of L-proline, L-ornithine and L-arginine. Previously, we reported cloning and characterization of two P5CS transcript variants generated by exon sliding that encode two protein isoforms differing only by a 2 amino acid-insert at the N-terminus of the γ-glutamyl kinase active site. The short form (P5CS.short) is highly expressed in the gut and is inhibited by ornithine. In contrast, the long form (P5CS.long) is expressed ubiquitously and is insensitive to ornithine. Interestingly, we found that all the established human cell lines we have studied expressed P5CS.long but not P5CS.short. In addition, expression of P5CS.long can be modulated by hormones: downregulation by hydrocortisone and dexamethasone and upregulation by estradiol, for example. Using a quantitative proteomic approach, we showed that P5CS.long is upregulated by p53 in p53-induced apoptosis in DLD-1 colorectal cancer cells. Functional genomic analysis confirmed that there are two p53-binding consensus sequences in the promoter region and in the intron 1 of the human P5CS gene. Interestingly, overexpression of P5CS by adenoviruses harboring P5CS.long or P5CS.short in various cell types has no effect on cell growth or survival. It would be of importance to investigate the role of P5CS as a p53 downstream effector and how P5CS.short expression is regulated by hormones and factors of alternative splicing in cells isolated from model animals.
Alternative splicing; Apoptosis; Exon sliding; Hormones; P53; Proline; Quantitative proteomics; Δ1-pyrroline-5-carboxylate (P5C); P5C synthase (P5CS)
The growth rate of several polyamine-deficient mutants of Escherichia coli was very low in minimal medium and increased markedly upon the addition of putrescine, spermidine, arginine, citrulline, or argininosuccinic acid. The endogenous content of polyamines was not significantly altered by the supplementation of polyamine-starved cultures with arginine or its precursors. In contrast, these compounds as well as putrescine or spermidine caused a 40-fold reduction in intracellular ornithine levels when added to polyamine-depleted bacteria. In vivo experiments with radioactive glutamic acid as a precursor and in vitro assays of the related enzymes showed that the decrease in ornithine levels was due to the inhibition of its biosynthesis rather than to an increase in its conversion to citrulline or delta 1-pyrroline-5-carboxylic acid and proline. High endogenous concentrations of ornithine were toxic for the E. coli strains tested. The described results indicate that the stimulatory effect of putrescine and spermidine on the growth of certain polyamine-starved bacteria may be partially due to the control of ornithine biosynthesis by polyamines.
This review deals with biochemical and physiological aspects of plant ornithine d-aminotransferase (OAT, EC 220.127.116.11). OAT is a mitochondrial enzyme containing pyridoxal-5′-phosphate as a cofactor, which catalyzes the conversion of L-ornithine to L-glutamate γ-semialdehyde using 2-oxoglutarate as a terminal amino group acceptor. It has been described in humans, animals, insects, plants and microorganisms. Based on the crystal structure of human OAT, both substrate binding and reaction mechanism of the enzyme are well understood. OAT shows a large structural and mechanistic similarity to other enzymes from the subgroup III of aminotransferases, which transfer an amino group from a carbon atom that does not carry a carboxyl function. In plants, the enzyme has been implicated in proline biosynthesis and accumulation (via pyrroline-5-carboxylate), which represents a way to regulate cellular osmolarity in response to osmotic stress. However, the exact metabolic pathway involving OAT remains a subject of controversy.
ornithine δ-aminotransferase; osmotic stress; proline; Δ1-pyrroline-5-carboxylate; pyridoxal-5′-phosphate; semialdehyde; transamination
Like many other plant species, Arabidopsis uses arginine (Arg) as a storage and transport form of nitrogen, and proline (Pro) as a compatible solute in the defence against abiotic stresses causing water deprivation. Arg catabolism produces ornithine (Orn) inside mitochondria, which was discussed controversially as a precursor for Pro biosynthesis, alternative to glutamate (Glu).
We show here that ornithine-δ-aminotransferase (δOAT, At5g46180), the enzyme converting Orn to pyrroline-5-carboxylate (P5C), is localised in mitochondria and is essential for Arg catabolism. Wildtype plants could readily catabolise supplied Arg and Orn and were able to use these amino acids as the only nitrogen source. Deletion mutants of δOAT, however, accumulated urea cycle intermediates when fed with Arg or Orn and were not able to utilize nitrogen provided as Arg or Orn. Utilisation of urea and stress induced Pro accumulation were not affected in T-DNA insertion mutants with a complete loss of δOAT expression.
Our findings indicate that δOAT feeds P5C exclusively into the catabolic branch of Pro metabolism, which yields Glu as an end product. Conversion of Orn to Glu is an essential route for recovery of nitrogen stored or transported as Arg. Pro biosynthesis occurs predominantly or exclusively via the Glu pathway in Arabidopsis and does not depend on Glu produced by Arg and Orn catabolism.
The enzymes in the arginine breakdown pathway (arginase, ornithine-δ-transaminase, and Δ′-pyrroline-5-carboxylate dehydrogenase) were found to be present in Bacillus licheniformis cells during exponential growth on glutamate. These enzymes could be coincidentally induced by arginine or ornithine to a very high level and their synthesis could be repressed by the addition of glucose, clearly demonstrating catabolite repression control of the arginine degradative pathway. The strongest catabolite repression control of arginase occurred when cells were grown on glucose and this control decreased when cells were grown on glycerol, acetate, pyruvate, or glutamate. The proline catabolite pathway was present in B. licheniformis during exponential growth on glutamate. The proline oxidation and the Δ′-pyrroline-5-carboxylate dehydrogenase in this breakdown pathway were induced by l-proline to a high level. The Δ′-pyrroline-5-carboxylate dehydrogenase was found to be under catabolite repression control. Arginase could be induced by proline and arginine addition induced proline oxidation, suggesting a common in vivo inducer for these convergent pathways.
We present a study of the enzymatic activities involved in the pathway for arginine catabolism by Agrobacterium tumefaciens. Nitrogen from arginine is recovered through the arginase-urease pathway; the genes for these two activities are probably chromosomally born. Arginase was found to be inducible during growth in the presence of arginine or ornithine. Urease was constitutively expressed. Ornithine, resulting from the action of arginase on arginine, could be used as a nitrogen source via transamination to delta 1-pyrroline-5-carboxylate and reduction of the latter compound to proline by a reductase (both enzymatic activities are probably chromosomally encoded). Ornithine could also be used as a carbon source. Thus, we identified an ornithine cyclase activity that was responsible for direct conversion of ornithine to proline. This activity was found to be Ti plasmid encoded and inducible by growth in medium containing octopine or nopaline. The same activity was also chromosomally encoded in some Agrobacterium strains. In such strains, this activity was inducible during growth in arginine-containing medium.
The soil bacterium Corynebacterium glutamicum, best known for its glutamate producing ability, is suitable as a producer of a variety of bioproducts. Glutamate is the precursor of the amino acid proline. Proline biosynthesis typically involves three enzymes and a spontaneous cyclisation reaction. Alternatively, proline can be synthesised from ornithine, an intermediate of arginine biosynthesis. The direct conversion of ornithine to proline is catalysed by ornithine cyclodeaminase. An ornithine overproducing platform strain with deletions of argR and argF (ORN1) has been employed for production of derived compounds such as putrescine. By heterologous expression of ocd this platform strain can be engineered further for proline production.
Plasmid-based expression of ocd encoding the putative ornithine cyclodeaminase of C. glutamicum did not result in detectable proline accumulation in the culture medium. However, plasmid-based expression of ocd from Pseudomonas putida resulted in proline production with yields up to 0.31 ± 0.01 g proline/g glucose. Overexpression of the gene encoding a feedback-alleviated N-acetylglutamate kinase further increased proline production to 0.36 ± 0.01 g/g. In addition, feedback-alleviation of N-acetylglutamate kinase entailed growth-coupled production of proline and reduced the accumulation of by-products in the culture medium.
The product spectrum of the platform strain C. glutamicum ORN1 was expanded to include the amino acid L-proline. Upon further development of the ornithine overproducing platform strain, industrial production of amino acids of the glutamate family and derived bioproducts such as diamines might become within reach.
Amino Acid; Proline; Corynebacterium Glutamicum; Metabolic Engineering; OCD; ORN1; Ornithine; Ornithine Cyclodeaminase; Platform Strain; Diamine; Putrescine; N-acetylglutamate Kinase
The moderately halophilic bacterium Halobacillus halophilus copes with the salinity in its environment by the production of compatible solutes. At intermediate salinities of around 1 M NaCl, cells produce glutamate and glutamine in a chloride-dependent manner (S. H. Saum, J. F. Sydow, P. Palm, F. Pfeiffer, D. Oesterhelt, and V. Müller, J. Bacteriol. 188:6808-6815, 2006). Here, we report that H. halophilus switches its osmolyte strategy and produces proline as the dominant solute at higher salinities (2 to 3 M NaCl). The proline biosynthesis genes proH, proJ, and proA were identified. They form a transcriptional unit and encode the pyrroline-5-carboxylate reductase, the glutamate-5-kinase, and the glutamate-5-semialdehyde dehydrogenase, respectively, catalyzing proline biosynthesis from glutamate. Expression of the genes was clearly salinity dependent and reached a maximum at 2.5 M NaCl, indicating that the pro operon is involved in salinity-induced proline biosynthesis. To address the role of anions in the process of pro gene activation and proline biosynthesis, we used a cell suspension system. Chloride salts lead to the highest accumulation of proline. Interestingly, chloride could be substituted to a large extent by glutamate salts. This unexpected finding was further analyzed on the transcriptional level. The cellular mRNA levels of all three pro genes were increased up to 90-fold in the presence of glutamate. A titration revealed that a minimal concentration of 0.2 M glutamate already stimulated pro gene expression. These data demonstrate that the solute glutamate is involved in the switch of osmolyte strategy from glutamate to proline as the dominant compatible solute during the transition from moderate to high salinity.
Cells of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 supplemented with micromolar concentrations of l-[14C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of l-[14C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [14C]ornithine preceded that of [14C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with l-[14C]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 Δ1pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an argF (ornithine carbamoyltransferase) insertional mutant was impaired in the production of [14C]citrulline from [14C]arginine; (ii) a proC (Δ1pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [14C]proline, [14C]glutamate, and [14C]glutamine from [14C]arginine or [14C]ornithine; and (iii) a putA (proline oxidase) insertional mutant did not produce [14C]glutamate from l-[14C]arginine, l-[14C]ornithine, or l-[14C]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 Δ1pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [14C]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.
Bacillus subtilis is known to accumulate large amounts of the compatible solute proline via de novo synthesis as a stress protectant when it faces high-salinity environments. We elucidated the genetic determinants required for the osmoadaptive proline production from the precursor glutamate. This proline biosynthesis route relies on the proJ-encoded γ-glutamyl kinase, the proA-encoded γ-glutamyl phosphate reductase, and the proH-encoded Δ1-pyrroline-5-caboxylate reductase. Disruption of the proHJ operon abolished osmoadaptive proline production and strongly impaired the ability of B. subtilis to cope with high-osmolarity growth conditions. Disruption of the proA gene also abolished osmoadaptive proline biosynthesis but caused, in contrast to the disruption of proHJ, proline auxotrophy. Northern blot analysis demonstrated that the transcription of the proHJ operon is osmotically inducible, whereas that of the proBA operon is not. Reporter gene fusion studies showed that proHJ expression is rapidly induced upon an osmotic upshift. Increased expression is maintained as long as the osmotic stimulus persists and is sensitively linked to the prevalent osmolarity of the growth medium. Primer extension analysis revealed the osmotically controlled proHJ promoter, a promoter that resembles typical SigA-type promoters of B. subtilis. Deletion analysis of the proHJ promoter region identified a 126-bp DNA segment carrying all sequences required in cis for osmoregulated transcription. Our data disclose the presence of ProA-interlinked anabolic and osmoadaptive proline biosynthetic routes in B. subtilis and demonstrate that the synthesis of the compatible solute proline is a central facet of the cellular defense to high-osmolarity surroundings for this soil bacterium.
The proline catabolic enzymes proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase catalyze the 4-electron oxidation of proline to glutamate. These enzymes play important roles in cellular redox control, superoxide generation, apoptosis and cancer. In some bacteria, the two enzymes are fused into the bifunctional enzyme, proline utilization A. Here we review the three-dimensional structural information that is currently available for proline catabolic enzymes. Crystal structures have been determined for bacterial monofunctional proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase, as well as the proline dehydrogenase and DNA-binding domains of proline utilization A. Some of the functional insights provided by analyses of these structures are discussed, including substrate recognition, catalytic mechanism, biochemical basis of inherited proline catabolic disorders and DNA recognition by proline utilization A.
Proline-Catabolism; Proline metabolism; Protein structure; X-ray crystallography; Proline dehydrogenase; P5C dehydrogenase; Proline utilization A; Ribbon-helix-helix
An enzyme system which converts ornithine to proline was partially purified from extracts of cells of Clostridium botulinum and of Clostridium PA 3670 by fractionation with ammonium sulfate and by dialysis in the presence of 0.01 m ornithine. Nicotinamide adenine dinucleotide (NAD) was the only cofactor required for maximal activity of the partially purified system. A possible intermediate in the conversion was accumulated when a high concentration of proline was used as substrate and the NAD was maintained in the oxidized state by adding lactic dehydrogenase. Small but significant amounts of this or a similar compound were trapped with either ornithine or proline as substrate when o-aminobenzaldehyde was added to reaction mixtures. The accumulation of the o-aminobenzaldehyde reaction product was NAD-dependent with both substrates. The compound accumulated from proline was identified as Δ1-pyrroline-5-carboxylic acid on the basis of the melting point of the 2,4-dinitrophenylhydrazone, and by paper chromatography of the reaction product formed with o-aminobenzaldehyde. Also, extracts of C. botulinum cells oxidized reduced NAD (NADH) in the presence of the product from proline or in the presence of Δ1-pyrroline-5-carboxylic acid, but did not do so in the presence of the other possible intermediate, Δ1-pyrroline-2-carboxylic acid. 14C-Δ1-pyrroline-5-carboxylic acid was reduced to 14C-proline by these extracts in the presence of NADH. These data indicate that the conversion of ornithine to proline by C. botulinum and Clostridium PA 3679 cells involves an oxidation of ornithine to glutamic-γ-semialdehyde which undergoes ring closure to Δ1-pyrroline-5-carboxylic acid. The latter compound is then reduced to proline.
Type II hyperprolinemia is an inherited abnormality in amino acid metabolism characterized by elevated plasma proline concentrations, iminoglycinuria, and the urinary excretion of delta1-pyrroline compounds. To define the enzymologic defect of this biochemical disorder, we developed a specific, sensitive radioisotopic assay for the proline degradative enzyme delta1-pyrroline-5-carboxylic acid dehydrogenase. Using this assay, we have shown an absence of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in the cultured fibroblasts from three patients with type II hyperprolinemia. We confirmed this result on cultured cells by demonstrating a similar absence of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in extracts prepared from the peripheral leukocytes of these patients. Additionally, we found significantly decreased levels of delta1-pyrroline-5-carboxylic acid dehydrogenase activity in the leukocyte extracts from five obligate heterozygotes for type II hyperprolinemia. We also demonstrated a reduction in leukocyte delta1-pyrroline-5-carboxylic acid dehydrogenase activity in three successive generations of a family. These results prove that an absence of delta1-pyrroline-5-carboxylic acid dehydrogenase is the enzymologic defect in type II hyperprolinemia and that this defect is inherited in an autosomal recessive fashion.
Proline is converted to glutamate in the yeast Saccharomyces cerevisiae by the sequential action of two enzymes, proline oxidase and delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase. The levels of these enzymes appear to be controlled by the amount of proline in the cell. The capacity to transport proline is greatest when the cell is grown on poor nitrogen sources, such as proline or urea. Mutants have been isolated which can no longer utilize proline as the sole source of nitrogen. Mutants in put1 are deficient in proline oxidase, and those in put2 lack P5C dehydrogenase. The put1 and put2 mutations are recessive, segregate 2:2 in tetrads, and appear to be unlinked to one another. Proline induces both proline oxidase and P5C dehydrogenase. The arginine-degradative pathway intersects the proline-degradative pathway at P5C. The P5C formed from the breakdown of arginine or ornithine can induce both proline-degradative enzymes by virtue of its conversion to proline.
Proline has long been known to accumulate in plants experiencing water limitation and this has driven studies of proline as a beneficial solute allowing plants to increase cellular osmolarity during water limitation. Proline metabolism also has roles in redox buffering and energy transfer and is involved in plant pathogen interaction and programmed cell death. Some of these unique roles of proline depend on the properties of proline itself, whereas others depend on the “proline cycle” of coordinated proline synthesis in the chloroplast and cytoplasm with proline catabolism in the mitochondria. The regulatory mechanisms controlling proline metabolism, intercellular and intracellular transport and connections of proline to other metabolic pathways are all important to the in vivo functions of proline metabolism. Connections of proline metabolism to the oxidative pentose phosphate pathway and glutamate-glutamine metabolism are of particular interest. The N-acetyl glutamate pathway can also produce ornithine and, potentially, proline but its role and activity are unclear. Use of model systems such as Arabidopsis thaliana to better understand both these long studied and newly emerging functions of proline can help in the design of next-generation experiments testing whether proline metabolism is a promising metabolic engineering target for improving stress resistance of economically important plants.
Human clinical isolates of Staphylococcus aureus, for example, strains Newman and N315, cannot grow in the absence of proline, albeit their sequenced genomes harbor genes for two redundant proline synthesis pathways. We show here that under selective pressure, S. aureus Newman generates proline-prototrophic variants at a frequency of 3 × 10−6, introducing frameshift and missense mutations in ccpA or IS1811 insertions in ptsH, two regulatory genes that carry out carbon catabolite repression (CCR) in staphylococci and other Gram-positive bacteria. S. aureus Newman variants with mutations in rocF (arginase), rocD (ornithine aminotransferase), and proC (Δ1-pyrroline 5-carboxylate [P5C] reductase) are unable to generate proline-prototrophic variants, whereas a variant with a mutation in ocd (ornithine cyclodeaminase) is unaffected. Transposon insertion in ccpA also restored proline prototrophy. CcpA was shown to repress transcription of rocF and rocD, encoding the first two enzymes, but not of proC, encoding the third and final enzyme in the P5C reductase pathway. CcpA bound to the upstream regions of rocF and rocD but not to that of proC. CcpA's binding to the upstream regions was greatly enhanced by phosphorylated HPr. The CCR-mediated proline auxotrophy was lifted when nonpreferred carbohydrates were used as the sole carbon source. The ccpA mutant displayed reduced staphylococcal load and replication in a murine model of staphylococcal abscess formation, indicating that carbon catabolite repression presents an important pathogenesis strategy of S. aureus infections.
A mutation resulting in inducer-independent expression of the proline-degradative enzymes was isolated in the yeast Saccharomyces cerevisiae. Strains carrying the mutation, put3, are partially constitutive for proline oxidase and delta 1-pyrroline-5-carboxylate dehydrogenase when grown on a medium lacking proline and are hyperinducible for both enzyme activities when grown on a proline-containing medium. put3 segregates as a single nuclear gene, is not linked to either of the presumed structural genes for proline oxidase and delta 1-pyrroline-5-carboxylate dehydrogenase, and does not affect proline transport. When heterozygous in diploid strains, put3 behaves neither fully dominant nor fully recessive. Endogenous induction by proline has been eliminated as a cause of the inducer-independent enzyme expression in the put3 mutant and the mutation is believed to be in a regulatory component of the proline-degradative pathway.