Repurposing Lipoic Acid Changes Electron Flow in Two Important
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Repurposing lipoic acid changes electron flow in two important metabolic pathways of Escherichia coli Morgan Anne Feeneya, Karthik Veeravallib, Dana Boyda, Stéphanie Gona,1, Melinda Jo Faulknera,2, George Georgioub, and Jonathan Beckwitha,3 aDepartment of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115; and bDepartments of Chemical Engineering, Molecular Genetics and Microbiology, and Institute for Cell and Molecular Biology, University of Texas, Austin, TX 78712 Contributed by Jonathan Beckwith, April 5, 2011 (sent for review February 25, 2011) In bacteria, cysteines of cytoplasmic proteins, including the essen- then can be reduced by the weak reductant, glutaredoxin 3, in- tial enzyme ribonucleotide reductase (RNR), are maintained in the dicating the key relevance of RNR to the growth defect (2, 3). reduced state by the thioredoxin and glutathione/glutaredoxin We have previously exploited the essentiality of RNR re- pathways. An Escherichia coli mutant lacking both glutathione re- duction to select for mutations that suppress the growth defect of ductase and thioredoxin reductase cannot grow because RNR is strains defective in both the thioredoxin and glutathione/gluta- disulfide bonded and nonfunctional. Here we report that suppres- redoxin pathways. For example, when the DTT required for sor mutations in the lpdA gene, which encodes the oxidative en- growth of a strain that lacks TrxB and Gor is removed, suppressor zyme lipoamide dehydrogenase required for tricarboxylic acid mutations allowing growth arise at a high frequency. These sup- (TCA) cycle functioning, restore growth to this redox-defective pressors carry mutations in the gene ahpC, which encodes a per- ahpC mutant. The suppressor mutations reduce LpdA activity, causing oxidase (peroxiredoxin), AhpC. The mutations in altered the substrate specificity of the enzyme, converting it from a per- the accumulation of dihydrolipoamide, the reduced protein-bound oxidase to a disulfide reductase, and restoring electron flow to the form of lipoic acid. Dihydrolipoamide can then provide electrons for glutathione/glutaredoxin pathway (4, 5). the reactivation of RNR through reduction of glutaredoxins. Dihy- We wished to determine whether pathways of electron transfer drolipoamide is oxidized in the process, restoring function to the to RNR in addition to the AhpC enzyme described above could be TCA cycle. Thus, two electron transfer pathways are rewired to evolved in E. coli. However, the frequency of suppressor muta- meet both oxidative and reductive needs of the cell: dihydrolipoa- tions in the ahpC gene was so high in the trxB gor strain that it MICROBIOLOGY mide functionally replaces glutathione, and the glutaredoxins re- masked other potential suppressor mutations. We report here the place LpdA. Both lipoic acid and glutaredoxins act in the reverse isolation of suppressor mutations of a trxB gor ahpCF triple-de- manner from their normal cellular functions. Bioinformatic analysis letion strain, in which no ahpC suppressor mutations can occur. suggests that such activities may also function in other bacteria. This new class of suppressor mutations all map to the gene for the oxidative enzyme lipoamide dehydrogenase, lpdA. Lipoic acid, disulfide bond | pyruvate dehydrogenase | α-ketoglutarate dehydrogenase | a small-molecule thiol-redox compound with a disulfide bond, bacterial genomes ordinarily acts as an oxidant; it is a cofactor for three multienzyme complexes in E. coli and is covalently attached to lysine residues eduction-oxidation reactions play key roles in many essential on proteins in these complexes in the form of lipoamide. Our Rmetabolic pathways. Some redox reactions involve the oxi- evidence indicates that these mutations suppress the redox defect dation or reduction of thiol residues either in an enzyme’s cys- by lowering the ability of LpdA to convert the reduced dihy- teines or in small redox-active molecules. In Escherichia coli, the drolipoamide to the oxidized lipoamide, leading to increased thiol-disulfide biology of the cell is compartmentalized; the ma- amounts of dihydrolipoamide in these protein complexes, which jority of protein thiols in the periplasm are oxidized (disulfide then serves as a source of electrons for the reduction of oxidized bonded), and the majority of protein thiols in the cytoplasm glutaredoxins. Further, it appears that in these suppressor strains, are reduced. However, for cytoplasmic enzymes that use cys- oxidized glutaredoxins are now required for a functional TCA teines in catalysis of reductive reactions, disulfide bonds do form, cycle because they oxidize dihydrolipoamide, providing a sub- albeit transiently. These bonds are rapidly reduced, restoring the stitute for the missing LpdA activity. In effect, this combination of enzyme’s activity. mutations results in alternative electron transfer steps for two essential intracellular pathways—that for reduction of ribonu- Two pathways maintain protein thiols in the reduced state in — the cytoplasm of E. coli (1). In the thioredoxin pathway, thio- cleotide reductase, and the oxidation steps in the TCA cycle redoxin 1 (encoded by trxA) and thioredoxin 2 (trxC) reduce ox- leading to a striking change in the fundamental redox biology of idized substrates. The resulting oxidized thioredoxins are then the cell. reduced by thioredoxin reductase (encoded by trxB) to regenerate Based on these studies, we suggest that there may be other their activity. In the glutathione/glutaredoxin pathway, gluta- bacterial species that use dihydrolipoamide as a source of cyto- redoxins 1 (grxA), 2 (grxB), and 3 (grxC) can reduce oxidized plasmic reducing power. Our genomic bioinformatic analyses of substrate proteins. The small molecule thiol glutathione (GSH) a large number of bacterial species indicate in which organisms and glutathione reductase (gor) provide electrons to maintain lipoamide/dihydrolipoamide might serve a function outside of its glutaredoxins in the reduced state. canonical roles in metabolism. The importance of the TrxB and Gor pathways is indicated by the finding that when null mutations in certain genes of both pathways are combined, E. coli cannot grow. The reason for this Author contributions: M.A.F., K.V., D.B., S.G., M.J.F., G.G., and J.B. designed research; M.A.F., synthetic lethality is that the essential enzyme ribonucleotide re- D.B., S.G., and M.J.F. performed research; M.A.F., K.V., D.B., G.G., and J.B. analyzed data; ductase (RNR) must be reduced by these pathways to maintain and M.A.F. and J.B. wrote the paper. its activity. In certain synthetic lethal combinations of mutations The authors declare no conflict of interest. (e.g., gor trxB), but not others (trxA trxC grxA), cell growth can be 1Present address: Centre d’Immunologie de Marseille-Luminy (CIML), Université de la restored by the addition of an exogenous reducing agent, such as Méditerranée, 13288 Marseille, France. DTT. The reducing agent restores electron flow into one or the 2Present address: Department of Microbiology, Cornell University, Ithaca, NY 14853. other of these defective thiol-redox pathways and in turn enables 3To whom correspondence should be addressed. E-mail: [email protected]. the reduction of RNR. The growth of the trxA trxC grxA mutant This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. strain can be restored simply by overexpression of RNR, which 1073/pnas.1105429108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1105429108 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Results Suppressor Mutations in lpdA Decrease Lipoamide Dehydrogenase Mutations in the Gene for Lipoamide Dehydrogenase Restore Growth Activity. lpdA encodes lipoamide dehydrogenase, an oxidative enzyme that plays a role in central metabolism as the E3 com- to a trxB, gor, ahpCF Mutant. To obtain novel suppressor mutations α of a strain lacking thioredoxin reductase (TrxB) and glutathione ponent of the pyruvate dehydrogenase and -ketoglutarate de- reductase (Gor), we used a triple mutant, SMG89, deleted for hydrogenase complexes, and as the L-protein of the glycine cleavage complex. LpdA oxidizes dihydrolipoamide moieties co- trxB, gor, and ahpCF. Construction of the triple-mutant strain, valently bound to lysine residues in proteins in these complexes, selection for suppressor mutations that restored growth, and thus regenerating active, oxidized lipoamide that has been re- mapping of the suppressor mutation are described in SI Materials duced in the process of oxidizing its substrates. Electrons are and Methods and Fig. S1. We mapped the suppressor mutation in transferred by LpdA from dihydrolipoamide to an FAD moiety one of the strains, SMG123, to the region containing a known bound to the enzyme, which, in turn, transfers electrons to NAD+. oxidoreductase, lpdA. Sequencing of lpdA in SMG123 revealed We mapped the amino acid alterations of LpdA that caused a point mutation that changed a conserved glycine residue into suppression onto the structure of a Pseudomonas putida homolog an aspartate (G186D). The five other suppressor strains from the of LpdA (6). All six amino acid changes altered amino acids that genetic selection also contained mutations in lpdA.Allsixmu- are close to or within the binding site of NAD+, the external tations (Gly183Cys, Gly183Ser, Ser164Phe, Ser164Tyr, Gly271Asp, oxidant of LpdA (Fig. 1). The FAD binding site is also close to and that of SMG123) changed small, uncharged amino acids into this region.