Dataset Information

Paradiplonema papillatum E3 interactome

ABSTRACT: In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a mitochondrial pyruvate dehydrogenase complex (PDH) to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) and the 2-oxoglutarate dehydrogenase (OGDH). In diplonemids, one of the most abundant and diverse groups of oceanic protists, comparative transcriptome analyses indicated that their PDH complex is compositionally unique, with the conventional E1 subunit replaced by an aceE protein of prokaryotic origin. Here we endogenously tagged the common E3 subunit with a protein A tag and performed immunoprecipitation experiments.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Diplonema Papillatum

SUBMITTER: Corinna Benz

LAB HEAD: Julius Lukes

PROVIDER: PXD035043 | Pride | 2023-07-17

REPOSITORIES: Pride

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Publications

Subunit composition of mitochondrial dehydrogenase complexes in diplonemid flagellates.

Záhonová Kristína K Valach Matus M Tripathi Pragya P Benz Corinna C Opperdoes Fred R FR Barath Peter P Lukáčová Veronika V Danchenko Maksym M Faktorová Drahomíra D Horváth Anton A Burger Gertraud G Lukeš Julius J Škodová-Sveráková Ingrid I

Biochimica et biophysica acta. General subjects 20230713 9

In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a tripartite mitochondrial pyruvate dehydrogenase (PDH) complex to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes with analogous composition catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) complex and the 2-oxoglutarate dehydrogenase (OGDH) complex. Comparative transcriptome ...[more]

PMID: 37451476

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Project description:Previous findings have demonstrated that the NADH/NAD+ ratio has a strong impact on the glycolytic flux in C. glutamicum under anaerobic conditions in the absence of external electron acceptors. During an attempt to rewire anaerobic metabolism to achieve high yield formation of ethanol, we inactivated the malate dehydrogenase and lactate dehydrogenase in a C. glutamicum strain expressing pyruvate decarboxylase and alcohol dehydrogenase, to eliminate formation of the by-products succinate and lactate, respectively. This modification increased the yield of ethanol but had a negative effect on glycolysis, which we found to correlate with an elevated NADH/NAD+ ratio. The pyruvate dehydrogenase (PDH) of C. glutamicum is active under anaerobic conditions, and can potentially exacerbate the negative effect on glycolysis, due to NADH formation. To reduce PDH activity under anaerobic conditions, we decided to replace the gene encoding the E3 subunit of PDH with its Escherichia coli counterpart, as E. coli PDH has been reported to be functional under aerobic conditions only. The resultant strain JS133 produced far less acetate with a further increased ethanol production, however, the glycolytic flux was still low. After observing differences in glycolytic flux for JS133 on glucose and fructose, we speculated that the pentose phosphate pathway (PPP) might be involved in the reduced flux on glucose. To prove this, we deleted the zwf gene, encoding glucose-6-phosphate dehydrogenase, which is the entry point into PPP, and immediately observed a stimulating effect on glycolysis. Subsequent characterization revealed a direct correlation between the intracellular NADH/NAD+ and NADPH/NADP+ ratios under anoxic conditions. Based on these findings we managed to re-channel the metabolism of C. glutamicum successfully towards either to ethanol or D-lactate with 92% and 98% of the theoretical yield respectively, which is the highest yields for D-lactate production thus far reported in the literature.

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	760-1.raw	Raw
	760-2.raw	Raw
	795-pdhd1.raw	Raw
	795-pdhd2.raw	Raw
	795-wtUV1.raw	Raw