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

ABSTRACT: 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.

SUBMITTER: Herzfeld A

PROVIDER: S-EPMC1164961 | biostudies-other | 1977 Jul

REPOSITORIES: biostudies-other

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Unraveling delta1-pyrroline-5-carboxylate-proline cycle in plants by uncoupled expression of proline oxidation enzymes.

Project description:The two-step oxidation of proline in all eukaryotes is performed at the inner mitochondrial membrane by the consecutive action of proline dehydrogenase (ProDH) that produces Delta(1)-pyrroline-5-carboxylate (P5C) and P5C dehydrogenase (P5CDH) that oxidizes P5C to glutamate. This catabolic route is down-regulated in plants during osmotic stress, allowing free Pro accumulation. We show here that overexpression of MsProDH in tobacco and Arabidopsis or impairment of P5C oxidation in the Arabidopsis p5cdh mutant did not change the cellular Pro to P5C ratio under ambient and osmotic stress conditions, indicating that P5C excess was reduced to Pro in a mitochondrial-cytosolic cycle. This cycle, involving ProDH and P5C reductase, exists in animal cells and now demonstrated in plants. As a part of the cycle, Pro oxidation by the ProDH-FAD complex delivers electrons to the electron transport chain. Hyperactivity of the cycle, e.g. when an excess of exogenous l-Pro is provided, generates mitochondrial reactive oxygen species (ROS) by delivering electrons to O(2), as demonstrated by the mitochondria-specific MitoSox staining of superoxide ions. Lack of P5CDH activity led to higher ROS production under dark and light conditions in the presence of Pro excess, as well as rendered plants hypersensitive to heat stress. Balancing mitochondrial ROS production during increased Pro oxidation is therefore critical for avoiding Pro-related toxic effects. Hence, normal oxidation of P5C to Glu by P5CDH is key to prevent P5C-Pro intensive cycling and avoid ROS production from electron run-off.

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Crystal structures of delta1-pyrroline-5-carboxylate reductase from human pathogens Neisseria meningitides and Streptococcus pyogenes.

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Project description:In plants, Δ1-pyrroline- 5-carboxylate synthase (P5CS) is the rate-limiting enzyme in proline biosynthesis. In this study, we introduced the LpP5CS (Lolium perenne L.) gene into switchgrass by Agrobacterium-mediated transformation. The transgenic lines (TG) were classified into two groups based on their phenotypes and proline levels. The group I lines (TG4 and TG6) had relatively high proline levels and improved biomass yield. The group II lines (TG1 and TG2) showed low proline levels, severely delayed flowering, stunted growth and reduced biomass yield. Additionally, we used RNA-seq analysis to detect the most significant molecular changes, and we analyzed differentially expressed genes, such as flowering-related and CYP450 family genes. Moreover, the biomass yield, physiological parameters, and expression levels of reactive oxygen species scavenger-related genes under salt stress all indicated that the group I plants exhibited significantly increased salt tolerance compared with that of the control plants, in contrast to the group II plants. Thus, genetic improvement of switchgrass by overexpressing LpP5CS to increase proline levels is feasible for increasing plant stress tolerance.

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Identification and characterization of D-hydroxyproline dehydrogenase and Delta1-pyrroline-4-hydroxy-2-carboxylate deaminase involved in novel L-hydroxyproline metabolism of bacteria: metabolic convergent evolution.

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Control, low water potential (drought) stress and stress release transcriptome analysis of mutants impaired in proline (Delta1-pyrroline-5-carboxylate synthase1-4; p5cs1-4) or abscisic acid (aba2-1) synthesis.

2016-08-05 | GSE75933 | GEO

Kinetics of human pyrroline-5-carboxylate reductase in L-thioproline metabolism.

Project description:L-Thioproline (L-thiazolidine-4-carboxylate, L-T4C) is a cyclic sulfur-containing analog of L-proline found in multiple kingdoms of life. The oxidation of L-T4C leads to L-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of L-T4C to L-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and L-Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with L-T4C using NAD(P)+ as the hydride acceptor. Both PYCRs exhibit significant L-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136 M-1 s-1) than PYCR1 (13.7 M-1 s-1). Interestingly, no activity was observed with either L-Pro or the analog DL-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize L-T4C as a substrate. Inhibition kinetics show that L-Pro is a competitive inhibitor of PYCR1 [Formula: see text] with respect to L-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that L-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of L-T4C.

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Project description:mRNA sequencing was used to identify genes differentially expressed in Delta1-pyrroline-5-carboxylate synthase1-4 (p5cs1-4) mutants deficient in low water potential-induced proline accumulation and the Abscisic Acid (ABA) deficient mutant aba2-1 compared to Col-0 wild type. Three treatments were analyzed for each genotype: unstressed control, a 96 hour low water potential (-1.2 MPa) treatment and a stress-release treatment where seedlings were removed from the stress treatment and transferred back to control media for 10 hours. Approximately 100 genes were found to be significantly up-regulated in p5cs1-4 compared to wild type in both the control and stress treatments with an equal number down regulated. There was also a substantial transcription response to the stress release treatment which varied between wild type, p5cs1-4 and aba2-1. Wild type (Col-0), p5cs1-4 and aba2-1 Arabidopsis thaliana seedlings were germinated on agar plates and after seven days of growth, seedlings were transferred either to fresh plates of control media or to low water potential (-1.2 MPa) stress plates. Samples were collected 96 h after transfer (Control and stress treatments). At 96 h, additional seedlings were transferred from the stress treatment back to control agar plates and samples collected 10 h after this stress release. Three independent biological experiments and mRNA extractions were performed for each genotype/treatment (27 mRNA extractions. After quality check and quantitation, the mRNA samples were pooled to produce a set of 9 samples for sequencing.

2016-08-05 | E-GEOD-75933 | biostudies-arrayexpress

Xenobiotic-Metabolizing Enzymes in Trematodes.

Project description:Trematode infections occur worldwide causing considerable deterioration of human health and placing a substantial financial burden on the livestock industry. The hundreds of millions of people afflicted with trematode infections rely entirely on only two drugs (praziquantel and triclabendazole) for treatment. An understanding of anthelmintic biotransformation pathways in parasites should clarify factors that can modulate therapeutic potency of anthelmintics currently in use and may lead to the discovery of synergistic compounds for combination treatments. Despite the pronounced epidemiological significance of trematodes, there is still no adequate understanding of the functionality of their metabolic systems, including xenobiotic-metabolizing enzymes. The review is focused on the structure and functional significance of the xenobiotic-metabolizing system in trematodes. Knowledge in this field can solve practical problems related to the search for new targets for antiparasitic therapy based on a focused action on certain elements of the parasite's metabolic system. Knowledge of the functionality of this system is required to understand the adaptation of the biochemical processes of parasites residing in the host and mechanisms of drug resistance development, as well as to select a promising molecular target for the discovery and development of new anthelmintic drugs.

| S-EPMC9775572 | biostudies-literature

Dietary n-6 PUFA deprivation downregulates arachidonate but upregulates docosahexaenoate metabolizing enzymes in rat brain.

Project description:BACKGROUND:Dietary n-3 polyunsaturated fatty acid (PUFA) deprivation increases expression of arachidonic acid (AA 20:4n-6)-selective cytosolic phospholipase A(2) (cPLA(2)) IVA and cyclooxygenase (COX)-2 in rat brain, while decreasing expression of docosahexaenoic acid (DHA 22:6n-3)-selective calcium-independent iPLA(2) VIA. Assuming that these enzyme changes represent brain homeostatic responses to deprivation, we hypothesized that dietary n-6 PUFA deprivation would produce changes in the opposite directions. METHODS:Brain expression of PUFA-metabolizing enzymes and their transcription factors was quantified in male rats fed an n-6 PUFA adequate or deficient diet for 15weeks post-weaning. RESULTS:The deficient compared with adequate diet increased brain mRNA, protein and activity of iPLA(2) VIA and 15-lipoxygenase (LOX), but decreased cPLA(2) IVA and COX-2 expression. The brain protein level of the iPLA(2) transcription factor SREBP-1 was elevated, while protein levels were decreased for AP-2? and NF-?B p65, cPLA(2) and COX-2 transcription factors, respectively. CONCLUSIONS:With dietary n-6 PUFA deprivation, rat brain PUFA metabolizing enzymes and some of their transcription factors change in a way that would homeostatically dampen reductions in brain n-6 PUFA concentrations and metabolism, while n-3 PUFA metabolizing enzyme expression is increased. The changes correspond to reported in vitro enzyme selectivities for AA compared with DHA.

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