Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development

Janet Storm, Jan Perner, Isabela Aparicio, Eva Maria Patzewitz, Kellen Olszewski, Manuel Llinas, Paul C. Engel, Sylke Müller

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Abstract

Background: Plasmodium falciparum contains three genes encoding potential glutamate dehydrogenases. The protein encoded by gdha has previously been biochemically and structurally characterized. It was suggested that it is important for the supply of reducing equivalents during intra-erythrocytic development of Plasmodium and, therefore, a suitable drug target. Methods. The gene encoding the NADP(H)-dependent GDHa has been disrupted by reverse genetics in P. falciparum and the effect on the antioxidant and metabolic capacities of the resulting mutant parasites was investigated. Results: No growth defect under low and elevated oxygen tension, no up- or down-regulation of a number of antioxidant and NADP(H)-generating proteins or mRNAs and no increased levels of GSH were detected in the D10gdha parasite lines. Further, the fate of the carbon skeleton of [13C] labelled glutamine was assessed by metabolomic studies, revealing no differences in the labelling of -ketoglutarate and other TCA pathway intermediates between wild type and mutant parasites. Conclusions: First, the data support the conclusion that D10gdha parasites are not experiencing enhanced oxidative stress and that GDHa function may not be the provision of NADP(H) for reductive reactions. Second, the results imply that the cytosolic, NADP(H)-dependent GDHa protein is not involved in the oxidative deamination of glutamate but that the protein may play a role in ammonia assimilation as has been described for other NADP(H)-dependent GDH from plants and fungi. The lack of an obvious phenotype in the absence of GDHa may point to a regulatory role of the protein providing glutamate (as nitrogen storage molecule) in situations where the parasites experience a limiting supply of carbon sources and, therefore, under in vitro conditions the enzyme is unlikely to be of significant importance. The data imply that the protein is not a suitable target for future drug development against intra-erythrocytic parasite development.

Original languageEnglish (US)
Article number193
JournalMalaria journal
Volume10
DOIs
StatePublished - Jul 26 2011

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Glutamate Dehydrogenase
Plasmodium falciparum
NADP
Parasites
Pharmaceutical Preparations
Proteins
Glutamic Acid
Carbon
Antioxidants
Reverse Genetics
Deamination
Metabolomics
Plasmodium
Glutamine
Ammonia
Skeleton
Genes
Oxidative Stress
Fungi
Up-Regulation

All Science Journal Classification (ASJC) codes

  • Parasitology
  • Infectious Diseases

Cite this

Storm, Janet ; Perner, Jan ; Aparicio, Isabela ; Patzewitz, Eva Maria ; Olszewski, Kellen ; Llinas, Manuel ; Engel, Paul C. ; Müller, Sylke. / Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development. In: Malaria journal. 2011 ; Vol. 10.
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title = "Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development",
abstract = "Background: Plasmodium falciparum contains three genes encoding potential glutamate dehydrogenases. The protein encoded by gdha has previously been biochemically and structurally characterized. It was suggested that it is important for the supply of reducing equivalents during intra-erythrocytic development of Plasmodium and, therefore, a suitable drug target. Methods. The gene encoding the NADP(H)-dependent GDHa has been disrupted by reverse genetics in P. falciparum and the effect on the antioxidant and metabolic capacities of the resulting mutant parasites was investigated. Results: No growth defect under low and elevated oxygen tension, no up- or down-regulation of a number of antioxidant and NADP(H)-generating proteins or mRNAs and no increased levels of GSH were detected in the D10gdha parasite lines. Further, the fate of the carbon skeleton of [13C] labelled glutamine was assessed by metabolomic studies, revealing no differences in the labelling of -ketoglutarate and other TCA pathway intermediates between wild type and mutant parasites. Conclusions: First, the data support the conclusion that D10gdha parasites are not experiencing enhanced oxidative stress and that GDHa function may not be the provision of NADP(H) for reductive reactions. Second, the results imply that the cytosolic, NADP(H)-dependent GDHa protein is not involved in the oxidative deamination of glutamate but that the protein may play a role in ammonia assimilation as has been described for other NADP(H)-dependent GDH from plants and fungi. The lack of an obvious phenotype in the absence of GDHa may point to a regulatory role of the protein providing glutamate (as nitrogen storage molecule) in situations where the parasites experience a limiting supply of carbon sources and, therefore, under in vitro conditions the enzyme is unlikely to be of significant importance. The data imply that the protein is not a suitable target for future drug development against intra-erythrocytic parasite development.",
author = "Janet Storm and Jan Perner and Isabela Aparicio and Patzewitz, {Eva Maria} and Kellen Olszewski and Manuel Llinas and Engel, {Paul C.} and Sylke M{\"u}ller",
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Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development. / Storm, Janet; Perner, Jan; Aparicio, Isabela; Patzewitz, Eva Maria; Olszewski, Kellen; Llinas, Manuel; Engel, Paul C.; Müller, Sylke.

In: Malaria journal, Vol. 10, 193, 26.07.2011.

Research output: Contribution to journalArticle

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T1 - Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development

AU - Storm, Janet

AU - Perner, Jan

AU - Aparicio, Isabela

AU - Patzewitz, Eva Maria

AU - Olszewski, Kellen

AU - Llinas, Manuel

AU - Engel, Paul C.

AU - Müller, Sylke

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AB - Background: Plasmodium falciparum contains three genes encoding potential glutamate dehydrogenases. The protein encoded by gdha has previously been biochemically and structurally characterized. It was suggested that it is important for the supply of reducing equivalents during intra-erythrocytic development of Plasmodium and, therefore, a suitable drug target. Methods. The gene encoding the NADP(H)-dependent GDHa has been disrupted by reverse genetics in P. falciparum and the effect on the antioxidant and metabolic capacities of the resulting mutant parasites was investigated. Results: No growth defect under low and elevated oxygen tension, no up- or down-regulation of a number of antioxidant and NADP(H)-generating proteins or mRNAs and no increased levels of GSH were detected in the D10gdha parasite lines. Further, the fate of the carbon skeleton of [13C] labelled glutamine was assessed by metabolomic studies, revealing no differences in the labelling of -ketoglutarate and other TCA pathway intermediates between wild type and mutant parasites. Conclusions: First, the data support the conclusion that D10gdha parasites are not experiencing enhanced oxidative stress and that GDHa function may not be the provision of NADP(H) for reductive reactions. Second, the results imply that the cytosolic, NADP(H)-dependent GDHa protein is not involved in the oxidative deamination of glutamate but that the protein may play a role in ammonia assimilation as has been described for other NADP(H)-dependent GDH from plants and fungi. The lack of an obvious phenotype in the absence of GDHa may point to a regulatory role of the protein providing glutamate (as nitrogen storage molecule) in situations where the parasites experience a limiting supply of carbon sources and, therefore, under in vitro conditions the enzyme is unlikely to be of significant importance. The data imply that the protein is not a suitable target for future drug development against intra-erythrocytic parasite development.

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