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Repurposing lipoic acid changes electron flow in two important metabolic pathways of Escherichia coli.

ABSTRACT: In bacteria, cysteines of cytoplasmic proteins, including the essential enzyme ribonucleotide reductase (RNR), are maintained in the reduced state by the thioredoxin and glutathione/glutaredoxin pathways. An Escherichia coli mutant lacking both glutathione reductase and thioredoxin reductase cannot grow because RNR is disulfide bonded and nonfunctional. Here we report that suppressor mutations in the lpdA gene, which encodes the oxidative enzyme lipoamide dehydrogenase required for tricarboxylic acid (TCA) cycle functioning, restore growth to this redox-defective mutant. The suppressor mutations reduce LpdA activity, causing the accumulation of dihydrolipoamide, the reduced protein-bound form of lipoic acid. Dihydrolipoamide can then provide electrons for the reactivation of RNR through reduction of glutaredoxins. Dihydrolipoamide is oxidized in the process, restoring function to the TCA cycle. Thus, two electron transfer pathways are rewired to meet both oxidative and reductive needs of the cell: dihydrolipoamide functionally replaces glutathione, and the glutaredoxins replace LpdA. Both lipoic acid and glutaredoxins act in the reverse manner from their normal cellular functions. Bioinformatic analysis suggests that such activities may also function in other bacteria.

SUBMITTER: Feeney MA

PROVIDER: S-EPMC3093452 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Repurposing lipoic acid changes electron flow in two important metabolic pathways of Escherichia coli.

Feeney Morgan Anne MA Veeravalli Karthik K Boyd Dana D Gon Stéphanie S Faulkner Melinda Jo MJ Georgiou George G Beckwith Jonathan J

Proceedings of the National Academy of Sciences of the United States of America 20110426 19

In bacteria, cysteines of cytoplasmic proteins, including the essential enzyme ribonucleotide reductase (RNR), are maintained in the reduced state by the thioredoxin and glutathione/glutaredoxin pathways. An Escherichia coli mutant lacking both glutathione reductase and thioredoxin reductase cannot grow because RNR is disulfide bonded and nonfunctional. Here we report that suppressor mutations in the lpdA gene, which encodes the oxidative enzyme lipoamide dehydrogenase required for tricarboxylic ...[more]

PMID: 21521794

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Project description:Understanding the allocation of the cellular proteome to different cellular processes is central to unraveling the organizing principles of bacterial physiology. Proteome allocation to protein translation itself is maximally efficient, i.e., it represents the minimal allocation of dry mass able to sustain the observed protein production rate. In contrast, recent studies on bacteria have demonstrated that the concentrations of many proteins exceed the minimal level required to support the observed growth rate, indicating some heterogeneity across pathways in their proteome efficiency. Here, we systematically analyze the proteome efficiency of metabolic pathways, which together account for more than half of the Escherichia coli proteome during exponential growth. Comparing the predicted minimal and the observed proteome allocation to different metabolic pathways across growth conditions, we find that the protein abundance in the most costly biosynthesis pathways-those for amino acid biosynthesis and cofactor biosynthesis-is regulated for near-optimal efficiency. Overall, proteome efficiency increases along the carbon flow through the metabolic network; proteins involved in pathways of nutrient uptake and central metabolism tend to be highly over-abundant, while proteins involved in anabolic pathways and in protein translation are much closer to the expected minimal abundance across conditions. Our work thus provides a bird's-eye view of metabolic pathway efficiency, demonstrating systematic deviations from optimal cellular efficiency at the network level. IMPORTANCE Protein translation is the most expensive cellular process in fast-growing bacteria, and efficient proteome usage should thus be under strong natural selection. However, recent studies show that a considerable part of the proteome is unneeded for instantaneous cell growth in Escherichia coli. We still lack a systematic understanding of how this excess proteome is distributed across different pathways as a function of the growth conditions. We estimated the minimal required proteome across growth conditions in E. coli and compared the predictions with experimental data. We found that the proteome allocated to the most expensive internal pathways, including translation and the synthesis of amino acids and cofactors, is near the minimally required levels. In contrast, transporters and central carbon metabolism show much higher proteome levels than the predicted minimal abundance. Our analyses show that the proteome fraction unneeded for instantaneous cell growth decreases along the nutrient flow in E. coli.

Project description:The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni²⁺ (Ni²⁺-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO₄²⁻ ions could restore hydrogen production to BL21(DE3); however, to only 25-30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO₄²⁻ were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO₄²⁻ and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis.

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Repurposing lipoic acid changes electron flow in two important metabolic pathways of Escherichia coli.

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Repurposing lipoic acid changes electron flow in two important metabolic pathways of Escherichia coli.

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