Publications

• Purified human erythrocyte pyrroline-5-carboxylate reductase. Preferential oxidation of NADPH.

  Merrill M.J., Yeh G.C., Phang J.M.

  Pyrroline-5-carboxylate reductase catalyzes the final step in proline synthesis by NAD(P)H-dependent reduction of pyrroline-5-carboxylate. We have purified and characterized this enzyme from human erythrocytes. Purification to homogeneity (approximately 600,000-fold) was accomplished by sonication ... >> More

  Pyrroline-5-carboxylate reductase catalyzes the final step in proline synthesis by NAD(P)H-dependent reduction of pyrroline-5-carboxylate. We have purified and characterized this enzyme from human erythrocytes. Purification to homogeneity (approximately 600,000-fold) was accomplished by sonication, ultracentrifugation, 2',5'-ADP-Sepharose affinity chromatography, and DEAE-Sephacel ion exchange chromatography. The enzyme runs as a single band of 30,000 Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sizing chromatography under nondenaturating conditions demonstrates activity in the 300,000-350,000 Mr range, suggesting that the native enzyme exists as a 10-to 12-mer. The purified enzyme exhibits kinetic characteristics similar to those previously described for whole red cell homogenates. The Vmax is 10-fold higher and the Km for pyrroline-5-carboxylate is 7-fold higher with NADH versus NADPH as cofactor. The affinity for NADPH is 15-fold higher than that for NADH. Erythrocyte pyrroline-5-carboxylate reductase is competitively inhibited by NADP+. Unlike the enzyme from some other sources, erythrocyte pyrroline-5-carboxylate reductase is not inhibited by proline or ATP. Double label studies using [14C]pyrroline-5-carboxylate and [3H]exNADPH in the presence of both NADH and NADPH were performed to determine the preferred source of reducing equivalents. In the presence of physiologic concentrations of pyrroline-5-carboxylate and both pyridine nucleotides, all of the reducing equivalents came from NADPH. We suggest that, in some cell types including human erythrocytes, a physiologic function of pyrroline-5-carboxylate reductase is the generation of NADP+. << Less

  J. Biol. Chem. 264:9352-9358(1989) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Deutch A.H., Smith C.J., Rushlow K.E., Kretschmer P.J.

  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 ... >> More

  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. << Less

  Nucleic Acids Res. 10:7701-7714(1982) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Functional specialization in proline biosynthesis of melanoma.

  De Ingeniis J., Ratnikov B., Richardson A.D., Scott D.A., Aza-Blanc P., De S.K., Kazanov M., Pellecchia M., Ronai Z., Osterman A.L., Smith J.W.

  Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then ... >> More

  Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of (13)C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis. << Less

  PLoS ONE 7:E45190-E45190(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Partial purification and some properties of delta1-pyrroline-5-carboxylate reductase from Escherichia coli.

  Rossi J.J., Vender J., Berg C.M., Coleman W.H.

  delta1-Pyrroline-5-carboxylate (PCA) reductase [L-proline:NAD(P)+5-oxidoreductase, EC 1.5.1.2] has been purified over 200-fold from Escherichia coli K-12. It has a molecular weight of approximately 320,000. PCA reductase mediates the pyridine nucleotide-linked reduction of PCA to proline but not t ... >> More

  delta1-Pyrroline-5-carboxylate (PCA) reductase [L-proline:NAD(P)+5-oxidoreductase, EC 1.5.1.2] has been purified over 200-fold from Escherichia coli K-12. It has a molecular weight of approximately 320,000. PCA reductase mediates the pyridine nucleotide-linked reduction of PCA to proline but not the reverse reaction (even at high substrate concentrations). The partially purified preparation is free of competing pyridine nucleotide oxidase, PCA dehydrogenase, and proline oxidase activities. The Michaelis constant (Km) values for the substrate, PCA, with reduced nicotinamide adenine dinucleotide phosphate (NADPH) or NADH as cofactor are 0.15 and 0.14 mM, respectively. The Km values determined for NADPH and NADH are 0.03 and 0.23 mM, respectively. Although either NADPH or NADH can function as cofactor, the activity observed with NADPH is severalfold greater. PCA reductase is not repressed by growth in the presence of proline, but it is inhibited by the reaction end products, proline and NADP. << Less

  J. Bacteriol. 129:108-114(1977) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Complementation of an Escherichia coli proC mutation by a gene cloned from Treponema pallidum.

  Gherardini F.C., Hobbs M.M., Stamm L.V., Bassford P.J. Jr.

  Little is known concerning the biosynthetic and metabolic capabilities of the syphilis agent, Treponema pallidum, because of the inability to cultivate continuously the organism in vitro. To circumvent the problem of cultivation, researchers have used recombinant DNA technology to express treponem ... >> More

  Little is known concerning the biosynthetic and metabolic capabilities of the syphilis agent, Treponema pallidum, because of the inability to cultivate continuously the organism in vitro. To circumvent the problem of cultivation, researchers have used recombinant DNA technology to express treponemal protein antigens in Escherichia coli. However, with a few notable exceptions, the specific cellular roles of these cloned treponemal proteins have not been determined. In this study, a cosmid library of T. pallidum genomic DNA was constructed and amplified by repackaging infective lambda bacteriophage particles in vivo. Recombinant clones capable of complementing a null mutation in the E. coli proC gene encoding 1-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2) were subsequently identified. The complementing activity was eventually localized to a 2.3-kilobase BglII-HindIII fragment that hybridized to the same-size fragment of a BglII-HindIII digest of T. pallidum DNA. Two proteins of 41 and 27 kilodaltons (kDa) were encoded by this fragment, as determined by maxicell analysis. Although only the 41-kDa protein could be specifically precipitated by experimental syphilitic rabbit antisera, it was the 27-kDa protein that was responsible for the proC-complementing activity. The recombinant P5C reductase differed from the native E. coli enzyme by a number of biochemical properties. The cloning of a T. pallidum gene encoding P5C reductase strongly suggests that this pathogen has the ability to synthesize proline and possibly other amino acids. << Less

  J. Bacteriol. 172:2996-3002(1990) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Pyrroline-5-carboxylate reductase in human erythrocytes.

  Yeh G.C., Harris S.C., Phang J.M.

  Pyrroline-5-carboxylate reductase, which converts pyrroline-5-carboxylate to proline, has been identified in human erythrocytes. The level of pyrroline-5-carboxylate reductase activity in these cells is comparable to the activity levels of major erythrocyte enzymes. The physiologic function of the ... >> More

  Pyrroline-5-carboxylate reductase, which converts pyrroline-5-carboxylate to proline, has been identified in human erythrocytes. The level of pyrroline-5-carboxylate reductase activity in these cells is comparable to the activity levels of major erythrocyte enzymes. The physiologic function of the enzyme in erythrocytes cannot be related to its function in other tissues, i.e., producing proline for protein synthesis. We examined the kinetic properties of erythrocyte pyrroline-5-carboxylate reductase and compared them to the properties of the enzyme from proliferating cultured human fibroblasts. We found that the kinetic properties and regulation of the erythrocyte enzyme are distinctly different from those for human fibroblast pyrroline-5-carboxylate reductase. These characteristics are consistent with the interpretation that the function of the enzyme in human erythrocytes may be to generate oxidizing potential in the form of NADP+. << Less

  J. Clin. Invest. 67:1042-1046(1981) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure, biochemistry, and gene expression patterns of the proline biosynthetic enzyme pyrroline-5-carboxylate reductase (PYCR), an emerging cancer therapy target.

  Bogner A.N., Stiers K.M., Tanner J.J.

  Proline metabolism features prominently in the unique metabolism of cancer cells. Proline biosynthetic genes are consistently upregulated in multiple cancers, while the proline catabolic enzyme proline dehydrogenase has dual, context-dependent pro-cancer and pro-apoptotic functions. Furthermore, t ... >> More

  Proline metabolism features prominently in the unique metabolism of cancer cells. Proline biosynthetic genes are consistently upregulated in multiple cancers, while the proline catabolic enzyme proline dehydrogenase has dual, context-dependent pro-cancer and pro-apoptotic functions. Furthermore, the cycling of proline and Δ¹-pyrroline-5-carboxylate through the proline cycle impacts cellular growth and death pathways by maintaining redox homeostasis between the cytosol and mitochondria. Here we focus on the last enzyme of proline biosynthesis, Δ¹-pyrroline-5-carboxylate reductase, known as PYCR in humans. PYCR catalyzes the NAD(P)H-dependent reduction of Δ¹-pyrroline-5-carboxylate to proline and forms the reductive half of the proline metabolic cycle. We review the research on the three-dimensional structure, biochemistry, inhibition, and cancer biology of PYCR. To provide a global view of PYCR gene upregulation in cancer, we mined RNA transcript databases to analyze differential gene expression in 28 cancer types. This analysis revealed strong, widespread upregulation of PYCR genes, especially PYCR1. Altogether, the research over the past 20 years makes a compelling case for PYCR as a cancer therapy target. We conclude with a discussion of some of the major challenges for the field, including developing isoform-specific inhibitors, elucidating the function of the long C-terminus of PYCR1/2, and characterizing the interactome of PYCR. << Less

  Amino Acids 53:1817-1834(2021) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Crystal structure of human pyrroline-5-carboxylate reductase.

  Meng Z., Lou Z., Liu Z., Li M., Zhao X., Bartlam M., Rao Z.

  Pyrroline-5-carboxylate reductase (P5CR) is a universal housekeeping enzyme that catalyzes the reduction of Delta(1)-pyrroline-5-carboxylate (P5C) to proline using NAD(P)H as the cofactor. The enzymatic cycle between P5C and proline is very important for the regulation of amino acid metabolism, in ... >> More

  Pyrroline-5-carboxylate reductase (P5CR) is a universal housekeeping enzyme that catalyzes the reduction of Delta(1)-pyrroline-5-carboxylate (P5C) to proline using NAD(P)H as the cofactor. The enzymatic cycle between P5C and proline is very important for the regulation of amino acid metabolism, intracellular redox potential, and apoptosis. Here, we present the 2.8 Angstroms resolution structure of the P5CR apo enzyme, its 3.1 Angstroms resolution ternary complex with NAD(P)H and substrate-analog. The refined structures demonstrate a decameric architecture with five homodimer subunits and ten catalytic sites arranged around a peripheral circular groove. Mutagenesis and kinetic studies reveal the pivotal roles of the dinucleotide-binding Rossmann motif and residue Glu221 in the human enzyme. Human P5CR is thermostable and the crystals were grown at 37 degrees C. The enzyme is implicated in oxidation of the anti-tumor drug thioproline. << Less

  J. Mol. Biol. 359:1364-1377(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Expression and kinetic characterization of PYCR3.

  Meeks K.R., Tanner J.J.

  PYCRs are proline biosynthetic enzymes that catalyze the NAD(P)H-dependent reduction of Δ¹-pyrroline-5-carboxylate (P5C) to proline in humans. PYCRs - especially PYCR1 - are upregulated in many types of cancers and have been implicated in the altered metabolism of cancer cells. Of the t ... >> More

  PYCRs are proline biosynthetic enzymes that catalyze the NAD(P)H-dependent reduction of Δ¹-pyrroline-5-carboxylate (P5C) to proline in humans. PYCRs - especially PYCR1 - are upregulated in many types of cancers and have been implicated in the altered metabolism of cancer cells. Of the three isoforms of PYCR, PYCR3 remains the least studied due in part to the lack of a robust recombinant expression. Herein, we describe a procedure for the expression of soluble SUMO-PYCR3 in Escherichia coli, purification of the fusion protein, and removal of the SUMO tag. PYCR3 is active with either NADPH or NADH as the coenzyme. Bi-substrate kinetic measurements obtained by varying the concentrations of both L-P5C and NADH, along with product inhibition data for l-proline, suggest a random ordered bi bi mechanism. A panel of 19 proline analogs was screened for inhibition, and the kinetics of competitive inhibition (with L-P5C) were measured for five of the compounds screened, including N-formyl-l-proline, a validated inhibitor of PYCR1. N-formyl-l-proline was found to be ten times more selective for PYCR1 over PYCR3. The SUMO-PYCR3 expression system should be useful for testing the isoform specificity of PYCR1 inhibitors. << Less

  Arch. Biochem. Biophys. 733:109468-109468(2023) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.