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Off-pathway, oxygen-dependent thiamine radical in the Krebs cycle.

ABSTRACT: The catalytic cofactor thiamine diphosphate is found in many enzymes of central metabolism and is essential in all extant forms of life. We demonstrate the presence of an oxygen-dependent free radical in the thiamine diphosphate-dependent Escherichia coli 2-oxoglutarate dehydrogenase, which is a key component of the tricarboxylic acid (Krebs) cycle. The radical was sufficiently long-lived to be trapped by freezing in liquid nitrogen, and its electronic structure was investigated by electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR). Taken together, the spectroscopic results revealed a delocalized pi radical on the enamine-thiazolium intermediate within the enzyme active site. The radical is generated as an intermediate during substrate turnover by a side reaction with molecular oxygen, resulting in the continuous production of reactive oxygen species under aerobic conditions. This off-pathway reaction may account for metabolic dysfunction associated with several neurodegenerative diseases. The possibility that the on-pathway reaction may proceed via a radical mechanism is discussed.

SUBMITTER: Frank RA

PROVIDER: S-EPMC7610923 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Off-pathway, oxygen-dependent thiamine radical in the Krebs cycle.

Frank René A W RA Kay Christopher W M CW Hirst Judy J Luisi Ben F BF

Journal of the American Chemical Society 20080110 5

The catalytic cofactor thiamine diphosphate is found in many enzymes of central metabolism and is essential in all extant forms of life. We demonstrate the presence of an oxygen-dependent free radical in the thiamine diphosphate-dependent Escherichia coli 2-oxoglutarate dehydrogenase, which is a key component of the tricarboxylic acid (Krebs) cycle. The radical was sufficiently long-lived to be trapped by freezing in liquid nitrogen, and its electronic structure was investigated by electron para ...[more]

PMID: 18183975

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Project description:BackgroundAlternol is a natural compound isolated from fermentation products of a mutant fungus. Our previous studies demonstrated that Alternol specifically kills cancer cells but spares benign cells.MethodsTo investigate the mechanism underlying alternol-induced cancer cell-specific killing effect, we took a comprehensive strategy to identify Alternol's protein targets in prostate cancer cells, including PC-3, C4-2, and 22RV1, plus benign BPH1 cell lines. Major experimental techniques included biotin-streptavidin pulldown assay coupled with mass-spectrometry, in vitro enzyme activity assay for Krebs cycle enzymes and gas chromatography-mass spectrometry (GC-MS) for metabolomic analysis.ResultsAmong 14 verified protein targets, four were Krebs cycle enzymes, fumarate hydratase (FH), malate dehydrogenase-2 (MDH2), dihydrolipoamide acetyltransferase (DLAT) in pyruvate dehydrogenase complex (PDHC) and dihydrolipoamide S-succinyltransferase (DLST) in a-ketoglutarate dehydrogenase complex (KGDHC). Functional assays revealed that PDHC and KGDHC activities at the basal level were significantly higher in prostate cancer cells compared to benign prostate BPH1 cells, while alternol treatment reduced their activities in cancer cells close to the levels in BPH1 cells. Although FH and MDH2 activities were comparable among prostate cancer and benign cell lines at the basal level, Alternol treatment largely increased their activities in cancer cells. Metabolomic analysis revealed that Alternol treatment remarkably reduced the levels of malic acid, fumaric acid, and isocitric acid and mitochondrial respiration in prostate cancer cells. Alternol also drastically reduced mitochondrial respiration and ATP production in PC-3 cells in vitro or in xenograft tissues but not in BPH1 cells or host liver tissues.ConclusionsAlternol interacts with multiple Krebs cycle enzymes, resulting in reduced mitochondrial respiration and ATP production in prostate cancer cells and xenograft tissues, providing a novel therapeutic strategy for prostate cancer treatment.

Dataset Information

Off-pathway, oxygen-dependent thiamine radical in the Krebs cycle.

Publications

Off-pathway, oxygen-dependent thiamine radical in the Krebs cycle.

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