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

ABSTRACT: 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.

SUBMITTER: Farres J

PROVIDER: S-EPMC1135432 | biostudies-other | 1988 Dec

REPOSITORIES: biostudies-other

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Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from <i>Mycobacterium tuberculosis:</i> Evidence for Substrate Channeling.

Project description:In Mycobacterium tuberculosis, proline dehydrogenase (PruB) and ∆1-pyrroline-5-carboxylate (P5C) dehydrogenase (PruA) are monofunctional enzymes that catalyze proline oxidation to glutamate via the intermediates P5C and L-glutamate-γ-semialdehyde. Both enzymes are essential for the replication of pathogenic M. tuberculosis. Highly active enzymes were expressed and purified using a Mycobacterium smegmatis expression system. The purified enzymes were characterized using natural substrates and chemically synthesized analogs. The structural requirements of the quinone electron acceptor were examined. PruB displayed activity with all tested lipoquinone analogs (naphthoquinone or benzoquinone). In PruB assays utilizing analogs of the native naphthoquinone [MK-9 (II-H2)] specificity constants Kcat/Km were an order of magnitude greater for the menaquinone analogs than the benzoquinone analogs. In addition, mycobacterial PruA was enzymatically characterized for the first time using exogenous chemically synthesized P5C. A Km value of 120 ± 0.015 µM was determined for P5C, while the Km value for NAD+ was shown to be 33 ± 4.3 µM. Furthermore, proline competitively inhibited PruA activity and coupled enzyme assays, suggesting that the recombinant purified monofunctional PruB and PruA enzymes of M. tuberculosis channel substrate likely increase metabolic flux and protect the bacterium from methylglyoxal toxicity.

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Structural determinants of oligomerization of ?(1)-pyrroline-5-carboxylate dehydrogenase: identification of a hexamerization hot spot.

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Project description:The emergence and rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) raise a critical need for alternative therapeutic options. New antibacterial drugs and targets are required to combat MRSA-associated infections. Based on this study, celastrol, a natural product from the roots of Tripterygium wilfordii Hook. f., effectively combats MRSA in vitro and in vivo. Multi-omics analysis suggests that the molecular mechanism of action of celastrol may be related to Δ1 -pyrroline-5-carboxylate dehydrogenase (P5CDH). By comparing the properties of wild-type and rocA-deficient MRSA strains, it is demonstrated that P5CDH, the second enzyme of the proline catabolism pathway, is a tentative new target for antibacterial agents. Using molecular docking, bio-layer interferometry, and enzyme activity assays, it is confirmed that celastrol can affect the function of P5CDH. Furthermore, it is found through site-directed protein mutagenesis that the Lys205 and Glu208 residues are key for celastrol binding to P5CDH. Finally, mechanistic studies show that celastrol induces oxidative stress and inhibits DNA synthesis by binding to P5CDH. The findings of this study indicate that celastrol is a promising lead compound and validate P5CDH as a potential target for the development of novel drugs against MRSA.

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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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Enhancement of Solubility, Purification, and Inclusion Body Refolding of Active Human Mitochondrial Aldehyde Dehydrogenase 2.

Project description:Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is predominantly linked with acetaldehyde detoxification in the second stage of alcohol metabolism. To intensively study ALDH2 function, a higher purity and uniform composition of the protein is required. An efficient Escherichia coli system for ALDH2 expression was developed by using His and a small ubiquitin-related modifier fusion tag. Most of the recombinant ALDH2s were expressed in the form of inclusion bodies. The ALDH2-enriched inclusion bodies were denatured with 6 M guanidine hydrochloride, and then ALDH2 was ultrafitrated. Finally, ALDH2 was successfully purified through affinity and gel filtration chromatography. The purified ALDH2 was finally preserved by the vacuum freeze-drying method, and its purity was determined to be higher than 95%, with a final media yield of 33.89 mg/L. The specific activity of ALDH2 was 6.1 × 104 U/mg. This work was the first to report pET-SUMO-ALDH2 recombinant plasmid expression in Escherichia coli, and the inclusion bodies were isolated and refolded. Finally, the purified ALDH2 had relatively higher purity, yield, and biological activity.

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Role of Δ1-pyrroline-5-carboxylate dehydrogenase supports mitochondrial metabolism and host-cell invasion of Trypanosoma cruzi.

Project description:Proline is crucial for energizing critical events throughout the life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease. The proline breakdown pathway consists of two oxidation steps, both of which produce reducing equivalents as follows: the conversion of proline to Δ(1)-pyrroline-5-carboxylate (P5C), and the subsequent conversion of P5C to glutamate. We have identified and characterized the Δ(1)-pyrroline-5-carboxylate dehydrogenase from T. cruzi (TcP5CDH) and report here on how this enzyme contributes to a central metabolic pathway in this parasite. Size-exclusion chromatography, two-dimensional gel electrophoresis, and small angle x-ray scattering analysis of TcP5CDH revealed an oligomeric state composed of two subunits of six protomers. TcP5CDH was found to complement a yeast strain deficient in PUT2 activity, confirming the enzyme's functional role; and the biochemical parameters (Km, kcat, and kcat/Km) of the recombinant TcP5CDH were determined, exhibiting values comparable with those from T. cruzi lysates. In addition, TcP5CDH exhibited mitochondrial staining during the main stages of the T. cruzi life cycle. mRNA and enzymatic activity levels indicated the up-regulation (6-fold change) of TcP5CDH during the infective stages of the parasite. The participation of P5C as an energy source was also demonstrated. Overall, we propose that this enzymatic step is crucial for the viability of both replicative and infective forms of T. cruzi.

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