Repurposing Lipoic Acid Changes Electron Flow in Two Important

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 reduction 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 | May 10, 2011 | vol. 108 | no. 19 | 7991–7996 Downloaded by guest on September 27, 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. The proximity of the amino acid changes isolated larger and/or charged residues (Fig. 1A). Gly271Asp and Ser164- as suppressor mutations to the cofactors essential for LpdA ac- Phe were each isolated in two independent selections. tivity led us to ask whether the suppressor mutations disrupted To confirm that mutations in lpdA were responsible for the LpdA function. suppression of the cytoplasmic redox defect, we cloned lpdAG186D Whereas wild-type E. coli can grow on minimal glucose, strains and lpdAG271D into a low-copy plasmid under the control of an that lack lpdA cannot; acetate and succinate are required to sup- isopropyl β-D-thiogalactopyranoside (IPTG)-inducible promoter. port the growth of this mutant on minimal glucose media (7). To However, neither of the plasmid-encoded mutant lpdA alleles assess the effect of the suppressor mutations on LpdA activity, we suppressed the growth defect of the trxB gor ahpC strain (Fig. 1C). transduced the mutations into an otherwise wild-type background Furthermore, expression of a plasmid-encoded wild-type copy of (DHB4) and tested for the ability of these strains to grow on lpdA abolished growth of the suppressor strain SMG123, sug- minimal glucose media. Like the lpdA null mutant, our suppressor gesting that the suppressor mutations were recessive and that they mutants in the wild-type background do not grow on minimal may have reduced LpdA activity. These results led us to ask glucose unless supplemented with acetate and succinate (Fig. 2A). whether a complete knockout of lpdA might also suppress the The phenotypic properties of these strains indicate that the suppressor mutations lower lipoamide dehydrogenase activity, redox defect of strain SMG89. We constructed a quadruple- which should result in the accumulation of reduced lipoamide trxB gor ahpC lpdA SI Materials and Methods deletion strain ( ; ). (dihydrolipoamide) to higher levels than wild-type cells. To de- This strain (MAF180), although it grew very poorly, did grow to temine whether this was the case in the trxB gor ahpCF lpdA some extent without the addition of an exogenous reductant suppressor strains, we measured the amounts of dihydrolipoamide (Fig. 1B), indicating weak suppression. The addition of (oxidized) covalently bound to a FLAG-tagged SucB, the E2 protein of the lipoic acid to the growth media did not improve the growth of the α-ketoglutarate dehydrogenase complex, using a direct sandwich quadruple mutant in liquid culture (Fig. 1B), but did improve ELISA. We did this by comparing the content of reduced thiols in growth on solid media as measured by larger colony size (Fig. S2). SucB in the wild-type and a suppressor strain. Because SucB Plasmid expression of lpdAG186D or lpdAG271D improved the contains no cysteine residues, free thiols detected could only be growth of the quadruple mutant containing the lpdA deletion due to the thiols in dihydrolipoamide. We detected the free thiols considerably, confirming that these mutant alleles of lpdA sup- by reacting them with a biotin-maleimide alkylating agent, which pressed the redox defect as long as the wild-type LpdA was not would only form covalent bonds with the reduced form of lip- present (Fig. 1C). oamide. Strikingly, we saw an 8- to 11-fold increase in the amount

Fig. 1. Mutations in the gene encoding lipoamide dehydrogenase suppress the trxB gor ahpCF mutant. (A) Crystal structure of the P. putida LpdA homolog showing the region surrounding the NAD+ cofactor (depicted in red) and the residues found mutated in the trxB gor ahpCF suppressor strains. (B) Growth of the suppressor strain SMG123 (trxB gor

ahpCF lpdAG186D) and the quadruple-mutant strain MAF180 (trxB gor ahpCF lpdA). Cultures were grown in triplicate, in NZ amine media (solid lines) or NZ amine media + lipoic acid (dashed lines) at 37 °C, and growth was followed by mea-

suring the increase in OD600 over 12 h. A representative curve is shown; the growth curve was repeated three times. (C) Expression of wild-type lpdA and two suppressor mutants,

lpdAG186D and lpdAG271D, cloned under the control of an IPTG-inducible promoter on a low copy-number plasmid. Strains carrying the indicated plasmids were streaked on rich NZ media containing spectinomycin to select for the plasmid and 1 mM IPTG, and were grown at 37 °C for 2 d.

7992 | www.pnas.org/cgi/doi/10.1073/pnas.1105429108 Feeney et al. Downloaded by guest on September 27, 2021 Fig. 2. A suppressor mutation in the lpdA gene reduces dihydrolipoamide oxidation. (A) Growth of strains with various lpdA alleles on minimal glucose media. Strains were streaked on minimal glucose media containing leucine and isoleucine, and grown at 37 °C for 1–2d.(B) Measurement of SucB bound

reduced dihydrolipoamide in DHB4 (wild-type) and the suppressor strain SMG123 (trxB gor ahpCF lpdAG186D). Total cellular protein isolated from stationary phase growing E. coli was treated with the thiol alkylating agent biotin-maleimide, and serial dilutions of the treated protein samples were added to plates coated with anti-FLAG antibody to speciﬁcally bind the SucB (encoding a C-terminal FLAG tag) protein. Biotin maleimide-thiol content in dihydrolipoamide bound to SucB was estimated by streptavidin-HRP treatment followed by monitoring A450.

of the reduced form of lipoic acid, dihydrolipoamide, bound to the Dihydrolipoamide Restores Electron Flow Through the Glutaredoxin SucB complex in SMG123 over that seen in the wild-type strain, Pathway. The evidence that lipoamide biosynthesis is required for DHB4 (Fig. 2B and Fig. S3). suppression and that dihydrolipoamide is present in the suppressor strain strengthened our hypothesis that it serves as a source of ﬁ ﬁ Dihydrolipoamide Is Required for Suppression. Our ndings indicate electrons for reduction of protein disul des in substrates such as MICROBIOLOGY that the suppressor mutations reduce LpdA activity, thus causing ribonucleotide reductase (RNR). Dihydrolipoamide has a low accumulation of the reduced form of lipoamide, dihydrolipoamide. redox potential (−290 mV)—even more reducing than glutathione This evidence raised the possibility that dihydrolipoamide was (−240 mV)—and is therefore a good potential source of reducing responsible for the suppression and acted as a reductant that power in the cell (9). Furthermore, in vitro studies have shown that compensated for the redox defect of the trxB gor ahpC strain. If this dihydrolipoamide can drive reduction of the glutaredoxins (10). were the case, the pathways for lipoamide biosynthesis should be We considered the possibility that dihydrolipoamide might act essential for suppression. analogously to glutathione in suppressor strains, serving as a There are two pathways by which lipoylated proteins are gen- source of reducing equivalents for intermediary proteins that can erated in E. coli, one using lipoic acid added to growth media reduce RNR, such as the glutaredoxins or the thioredoxins. and the other using endogenously synthesized lipoate. The lplA Therefore, we asked whether thioredoxins or glutaredoxins were pathway uses the lplA gene product to ligate imported lipoic acid required for suppression in the trxB gor ahpCF suppressor strain onto substrate proteins; the lipA-lipB pathway both synthesizes SMG123. The thioredoxins were not required; however, deletion endogenous lipoate and ligates it onto proteins (8). Neither of grxA (glutaredoxin 1) from SMG181 drastically reduced growth pathway is essential for the growth of wild-type E. coli on rich medium. However, we wished to determine whether these pathways had become essential for growth of the trxB gor ahpC Table 1. Requirements for suppression lpdA strain. G186D Deletion in suppressed trxB trxB not We constructed derivatives of the suppressor strain SMG123, which lacked either the lipAB or lplA pathway, but had a com- strain expressed expressed plementing plasmid (pBAD18-trxB) to allow for growth of the None ++ + mutant strains (SI Materials and Methods). The trxB gor ahpCF Lipoamide lipB +0 lpdAG186D lipA/pBAD18-trxB strain (MAF493) is unable to grow biosynthesis unless arabinose is added to induce expression of the com- lipA +0 plementing trxB (Table 1). The growth defect of strain MAF493 lplA ++ + was overcome by addition of lipoic acid to the growth media. The Lipoylated sucB +0 restoration of growth is presumably due to the presence of the proteins lplA pathway. Thus, we constructed versions of the suppressor gcvH +0 strain that lacked both pathways for lipoyl protein ligation (lipA ± lplA and lipB lplA mutant strains). Addition of exogenous lipoate Glutaredoxins grxA + to the growth media did not rescue the growth of either the lipA grxC ++ + lplA or lipB lplA strain when expression from the complementing grxA, grxC +0 pBAD18-trxB plasmid was shut off. Thioredoxins trxA ++ + These data suggest that dihydrolipoamide is required for sup- trxC ++ + pression. Our results indicate that each of the lipoylated proteins trxA, trxC ++ + in E. coli may be individually required for suppression as well. We Deletions of genes involved in lipoamide biosynthesis, and of the constructed derivatives of the suppressor strain, SMG181, which glutaredoxins and thioredoxin genes, were transduced into SMG181 (the lacked the lipoylated proteins SucB and GcvH. These strains, like trxB gor ahpC lpdAsupp strain, SMG123, carrying a complementing plasmid, the version of the suppressor strain that lacked the genes for pBAD18-trxB) by P1 transduction. The resulting transductants were tested lipoamide biosynthesis, required arabinose (trxB induction) for for their ability to grow on NZ/ampicillin media with either 0.2% glucose (to growth (Table 1 and Fig. S4). We were unable to construct repress transcription of trxB) or with 0.2% arabinose (to induce trxB). ++, a version of SMG181 that lacked aceF, suggesting that it too may comparable growth to that of SMG123; +, smaller colonies; ±, very small be essential. colonies; 0, no colonies observed after 3 d of incubation.

Feeney et al. PNAS | May 10, 2011 | vol. 108 | no. 19 | 7993 Downloaded by guest on September 27, 2021 of the strain (MAF502). There was a small amount of growth in the strain carrying the deletion of grxA (tiny colonies after 3 d growth on rich media at 37 °C). Because grxC is up-regulated under conditions of oxidative stress, and because it is known to be reduced by dihydrolipoamide in vitro (10), we asked whether grxC was responsible for the residual growth of MAF502 by intro- ducing deletions of both grxA and grxC into the suppressor strain. This strain failed to grow without expression of trxB from the complementing plasmid, indicating that the glutaredoxins are required for suppression (Table 1 and Fig. S4).

Oxidation of Dihydrolipoamide by the Glutaredoxins. The indication that dihydrolipoamide provides electrons for the reduction of RNR in the suppressor strains also makes it likely that dihydrolipoamide itself becomes oxidized upon reducing whatever substrate it acts on (likely the glutaredoxins). If this were the case, the very activity of promoting the reduction of RNR (and other substrates) should allow the resultant oxidized lipoamide to participate in the 2-oxoacid dehydrogenase reactions of the TCA cycle. Therefore, we predicted that the trxB gor ahpC lpdAsupp strain would be able to grow on minimal glucose media lacking acetate and succinate, even though it is lacking the activity of an enzyme, LpdA, ordinarily essential for oxidation in the TCA cycle. This prediction was fulfilled; whereas the mutant lpdA alleles in a wild-type background conferred a requirement for acetate and succinate on minimal glucose media, paradoxi- cally, the suppressor strains grew on minimal glucose showing no such requirement (Fig. 3A). Thus, at the same time that the lpdA mutations suppressed the growth defect of the trxB gor ahpC mutant, the alteration of the redox state of glutaredoxins in this Fig. 3. Glutaredoxins replace the function of LpdA in the oxidizing cyto- same background appears to have provided a source of oxidation plasm. (A) Strains were grown on M63 minimal glucose media (supple- of dihydrolipoamide, in effect, replacing LpdA (Fig. 3). mented with leucine and isoleucine) at 37 °C for 2 d. (B) Strains were grown These results suggested that the glutaredoxins had become on M63 minimal glucose media (supplemented with leucine and isoleucine) essential for the functioning of the TCA cycle in the suppressor at 37 °C for 2 d, with either 0.2% arabinose (Right) or 0.2% arabinose plus 5 strains. We therefore asked whether the trxB gor ahpC lpdA-supp mM acetate and 5 mM succinate (Left) for 3 d at 37 °C. Counterclockwise strain could grow on minimal glucose media when grxA and grxC from the top, the strains are: MAF259 (trxB gor ahpCF lpdAG186D/pBAD18- were deleted. On media containing arabinose, trxB is induced trxB), MAF501 (MAF259 grxC), MAF502 (MAF259 grxA), and MAF503 (MAF259 grxA grxC). (C) Proposed scheme for the oxidation of dihy- from the complementing plasmid, pBAD18-trxB, and the glu- drolipoamide and reduction of glutaredoxins in the suppressor strains. taredoxins are not required for the reduction of RNR. The suppressor strain should therefore be able to grow on minimal glucose plus arabinose, independently of the glutaredoxin pathways can exhibit phenotypes that anticipate subsequently discov- way. However, like an lpdA null strain, the trxB gor ahpC lpdAsupp ered novel redox biology in other organisms (12, 13). Therefore, grxA grxC strain grows only when acetate and succinate are added we were inspired to seek evidence that dihydrolipoamide might to supplement the TCA cycle. (Fig. 3B) reduce the glutaredoxins in other bacteria. It was already known fi that some organisms, such as certain archaea, possess a gene encod- Cytoplasmic Disul de Bond Formation in the Suppressor Strain. The ing a lipoamide dehydrogenase, but lack its usual substrates such cytoplasm is normally a reducing environment, where any disul- as the dehydrogenase complexes of the TCA cycle (14). Although fide bonds formed in proteins are reduced by thioredoxins or the role of LpdA homologs in these organisms is unknown, their glutaredoxins. However, in the absence of TrxB, the thioredoxins accumulate in the oxidized form and can oxidize substrate pro- existence led us to speculate that some organisms might use lip- teins that are derivatives of proteins missing their signal sequen- oamide dehydrogenase and lipoamide for novel functions, in- ces (11). Even more efficient production of disulfide-bonded cluding possibly the reductive pathways we have found in the trxB proteins occurs in a strain (SMG96) deleted for both the trxB and gor ahpC suppressor SMG123. gor genes and carrying a suppressor mutation in the ahpC gene We initiated a bioinformatic analysis of bacterial genomes to that restores growth (12). We asked whether the suppressor strain identify candidate organisms that might use dihydrolipoamide as fi a source of reduction for its glutaredoxins. In this search, we described here would also exhibit high levels of disul de bond fi formation in proteins. We introduced a plasmid encoding signal identi ed a list of organisms that lacked the capacity for gluta- sequenceless tissue plasminogen activator (vtPA), which has thione biosynthesis, but did possess the genes for glutaredoxins multiple disulfide bonds arranged in a complex pattern, as well as and lipoamide biosynthesis (Table S1). Some of the organisms a plasmid encoding a signal sequenceless disulfide isomerase identified in our search are those that might be expected to use (DsbC). We found that there is about a 119.4 ± 10.9 (n = 3) in- other thiol redox pathways, such as the Bacillus species, which crease in vtPA activity in SMG96 compared with a wild-type use bacillithiol (15), or the Actinobacteria, which use mycothiol strain (DHB4), whereas SMG123 exhibits an ∼234.4 ± 41.4.1 (16), small-molecule thiols analogous to glutathione. However, (n = 2) increase compared with DHB4 (approximately a twofold others may lack the thiol redox pathways and thus require a re- increase over the levels seen in SMG96). ductant for their glutaredoxins. Moreover, among these 40-some organisms, we identified a subset that also lacked the genes for Possible Alternative Roles of Lipoamide Dehydrogenase and Lipoamide the E2 components of the dehydrogenase complexes (Table 2). in Other Organisms. Our results suggest that lipoamide may be able These organisms presumably do not use lipoamide and dihy- to function outside of its canonical role in metabolism, at least in drolipoamide dehydrogenase for their canonical metabolic the suppressor strains described here. Past work has shown that functions, but instead may use (dihydro)lipoamide to carry out E. coli mutants selected for alterations of their thiol redox path- other redox reactions.

7994 | www.pnas.org/cgi/doi/10.1073/pnas.1105429108 Feeney et al. Downloaded by guest on September 27, 2021 In addition to identifying genomes that lacked E2 homologs, for enzymatic catalysis. There are many factors (competition, − we also found that two of the lip+ gsh grx+ genomes identified in specificity, accessibility, oxidation by LpdA or other oxidants, etc.) our bioinformatic search had LpdA homologs with an N-terminal that might have limited the use of the strong reductive power of lipoyl domain (Table 2). These LpdA homologs, in effect, could dihydrolipoamide in physiological pathways. Our findings show that contain their own substrate and thus have an oxidoreductase reduced dihydrolipoamide can serve as a source of electrons for key activity separable from the dehydrogenase complexes to which reactions in the cell. Because our results establish that these reac- LpdA normally belongs. Lipoyl domain-containing LpdA homo- tions can take place in vivo, we asked whether there are organisms logs have been previously identified in bacteria such as Strepto- that might naturally take advantage of dihydrolipoamide as a coccus, Neisseria, and Clostridia, among others (17–20). The source of electron equivalents. Our bioinformatic analysis raises physiological role of the lipoyl-domain lipoamide dehydrogenases the possibility that the glutaredoxin/lipoamide coupling in vivo in remains to be elucidated, but given that their activity is separable E. coli may function in several other bacteria. from the dehydrogenase complexes, it may be that these proteins Several lines of evidence show that the lpdA suppressor can also function in pathways similar to the one that we have mutations are defective in lipoamide dehydrogenase (LpdA) described in this work. activity: (i) The mutants are all recessive to the wild-type allele of lpdA;(ii) The mutations, when transferred into a wild-type Discussion background, all behave phenotypically like lpdA null mutations; In this paper, we describe a remarkable shift in two electron (iii)InanlpdA suppressor strain (SMG123), direct assay of the transfer pathways important for cellular function. These two redox state of the lipoamide bound to one of the TCA cycle redesigned pathways are the result of a combination of muta- complexes shows a substantial increase in the amount of reduced tions that inactivate the reductive thioredoxin and glutathione/ lipoamide (dihydrolipoamide) compared with a wild-type strain. glutaredoxin pathways and, separately, alter the function of the The defects in LpdA activity prevent the oxidation of dihy- oxidative enzyme lipoamide dehydrogenase (LpdA). The lpdA drolipoamide to lipoamide, allowing dihydrolipoamide to accu- mutations were obtained by in vivo genetic selections in E. coli mulate in its reduced state; and (iv) All of the mutations cause trxB gor ahpC cells. As a consequence of these changes, (i) the changes in or very close to the NAD binding site of LpdA, in- source of electrons to reduce and maintain activity of such dicating that they could be interfering with electron flow between enzymes as ribonucleotide reductase is shifted from NADPH to NAD and the FAD bound to LpdA. dihydrolipoamide and (ii) the oxidative power to generate the oxidized lipoamide necessary for TCA cycle function is shifted

from NAD to oxidized glutaredoxins. In this way, the mutant MICROBIOLOGY cells have reversed the normal role of two key redox components, lipoic acid and glutaredoxin. There is currently widespread interest in engineering bacteria to carry out new tasks by gen- erating significant changes in metabolic pathways or by in- corporating new metabolic pathways (for examples, see refs. 21– 23). Our findings further emphasize the metabolic and genetic plasticity of electron flow pathways in bacteria. Although reduced lipoic acid is a powerful reductant, the known physiological functions of lipoic acid are oxidative. However, in vitro work has shown that dihydrolipoamide can serve as a source of electrons for the reduction of glutaredoxins (10) and for the transfer of electrons to the M. tuberculosis peroxidase AhpC (24) and the organic hydroperoxide resistance protein Ohr from Xylella fastidiosa (25). These in vitro studies leave open the question of whether reduced dihydrolipoamide can serve in vivo as an electron donor

Table 2. Bioinformatic analysis of organisms that could potentially use a novel pathway for dihydrolipoamide Genome Phylogeny Grx E2 LpdA

Chlorobium_tepidum_TLS Chlorobi 1 0 2 Desulfovibrio_vulgaris__Miyazaki_F_ Proteobacteria 1 0 2 Moorella_thermoacetica_ATCC_39073 Firmicutes 1 0 1 Thermoanaerobacter_X514 Firmicutes 1 1 2 Thermoanaerobacter_pseudethanolicus_ Firmicutes 1 1 2 ATCC_33223 Thermodesulfovibrio_yellowstonii_ Nitrospirae 1 0 1 DSM_11347 Fig. 4. Model for the rewired electron transfer pathways in the trxB gor ﬂ Treponema_denticola_ATCC_35405 Spirochaetes 1 0 1 ahpC lpdAsupp strain. Arrows represent the direction of electron ow. In wild- type cells (A), NADPH is used as a source of reducing power for the thio- The number of homologs of each gene is given in the columns; these redoxin and glutathione/glutaredoxin pathways, which reduce disulﬁde organisms lack gshA and the pathway for glutathione biosynthesis, but pos- bonds in substrate proteins such as RNR. The oxidative reaction of lipoamide sess proteins homologous to glutaredoxins (Grx). At the same time, they dehydrogenase, LpdA, is used to regenerate active, oxidized lipoamide for have the biosynthetic capacity for dihydrolipoamide biosynthesis, but ap- the function of the α-ketoglutarate and pyruvate dehydrogenases and the pear to lack homologs of the E2 proteins which in E. coli are lipoylated glycine cleavage complex. (B)InthetrxB gor ahpC lpdAsupp strains, LpdA is and function in the 2-oxoacid dehydrogenase complexes. Organisms high- inactive and dihydrolipoamide accumulates. The thioredoxin and gluta- lighted in gray possess the E2 homologs that participate in the dehydroge- redoxin pathways are inactive, and oxidized glutaredoxins accumulate. nase complexes, but also have a second homolog of LpdA that contains an N- These glutaredoxins oxidize dihydrolipoamide to lipoamide, allowing for the terminal lipoyl domain, and that could potentially act independently as an function of the multienzyme complexes, whereas the glutaredoxins are oxidoreductase. themselves reduced and able to go on to reduce substrate proteins.

Feeney et al. PNAS | May 10, 2011 | vol. 108 | no. 19 | 7995 Downloaded by guest on September 27, 2021 We have shown that when we introduce into the lpdA sup- strains seems to be largely due to the glutaredoxins, as the de- pressor strains mutations that lack the dihydrolipoamide bio- letion of both glutaredoxins abolishes TCA cycle function. synthetic pathway, the strains become dependent on exogenous Finally, we have shown that this class of suppressor strains is lipoic acid for growth. Further, the ability of the exogenous lipoic able to produce correctly folded, active disulfide bonded proteins acid to restore growth is dependent on lipoyl ligase (lplA). These in its cytoplasm and is potentially useful for recombinant protein results indicate that lipoic acid covalently bound to TCA cycle production. Because of slow growth, the use of the strain for this complexes is essential for the suppressor effect of the lpdA purpose is currently limited. However, addition of lipoic acid to mutations. We are led to conclude that the enzyme-bound dihy- the media gives the suppressor strain SMG123 a moderate in- drolipoamide has replaced glutathione as a source of electrons for crease in growth (Fig. 1C). Therefore, it may be possible to im- reduction of disulfide bonds in enzymes such as RNR. The de- prove the growth of this strain further, while maintaining efficient pendency of the suppression on glutaredoxins indicates that this fi reduction is accomplished by the transfer of electrons first from cytoplasmic disul de bond formation, by additional mutations or dihydrolipoamide to glutaredoxins and thence to RNR. Our by optimizing media conditions. results indicate that the reduction of glutaredoxins by dihy- Materials and Methods drolipoamide, which had been previously shown to occur in vitro (10), can occur in vivo to a physiologically significant extent. Strains and Growth Conditions. Strains and plasmids used in this work are Our conclusion that oxidized glutaredoxins can functionally listed in Table S2 and primers are listed in Table S3. All strains were grown on replace lipoamide dehydrogenase is based on the finding that rich media (NZ-amine) (26) at 37 °C unless specified otherwise. Additional though wild-type strains carrying the lpdA suppressor mutations details of growth conditions are in SI Materials and Methods. are unable to grow on minimal glucose media unless acetate and succinate are added, counterintuitively, the trxB gor ahpC strain Determination of Reduced Dihydrolipoamide Levels. Strains carrying the C- carrying the same lpdA mutants do grow on minimal media. Thus, terminally FLAG-tagged sucB were grown to stationary phase in LB media. the functioning of the TCA cycle, which is abrogated in wild-type After cells were lysed with a French press, cell debris was removed by cen- cells carrying the lpdA mutations, is restored in the trxB gor ahpC trifugation and the proteins concentrated using Millipore Ultra-4 Centrifugal mutant carrying the very same lpdA mutations. The rewiring of Filters (molecular weight cutoff 3,000). Biotin-maleimide (5 mM) was added these electron transfer pathways is shown in Fig. 4. to the protein concentrate and allowed to react with free thiols for 20–30 min Interestingly, the mutations we isolated as suppressors of the at 37 °C. Excess biotin-maleimide was removed using PD-10 desalting columns trxB gor ahpC strain were all missense mutations. This finding, (Millipore). Serial dilutions of these samples were then added to ELISA plates along with the result that a deletion of the lpdA gene acts only very that had been coated with anti-FLAG antibody, incubated at room temper- weakly as a suppressor, suggests that the restoration of growth to ature for 1 h, and washed three times with PBS Tween-20. Streptavidin-HRP the gor trxB ahpC strain by the lpdA suppressor mutations depends (Sigma) was added at 1:10,000 in PBS + 1% milk and incubated at room on the presence of an intact LpdA protein even though it is temperature for 1 h. After three washes with PBST, 75 μL ultra TMB was mutant. This property of the mutants could be because the added and the reaction allowed to proceed for 30 min before it was stopped μ presence of the LpdA protein is necessary to stabilize the struc- with 75 Lof2MH2SO4. Absorbance was then measured at 450 nm. ture of the dihydrolipoamide-containing multienzyme complexes or because a small amount of residual activity in the mutant ACKNOWLEDGMENTS. We thank J. Cronan and J. Imlay for kindly providing ’ proteins is necessary for suppression. Our results suggest that the strains used in this work. We also thank members of J.B. s laboratory for critical discussion and assistance. This work was supported by National Insti- latter explanation may be correct, as the expression of a catalyti- tutes of Health Grants GM41883 and GM55090 and by Human Frontier Sci- cally inactive lipoamide dehydrogenase does not suppress. How- ence Organization Grant CDA0034/2007. J.B. is an American Cancer Society ever, the oxidation of dihydrolipoamide in these suppressor Professor.

1. Ritz D, Beckwith J (2001) Roles of thiol-redox pathways in bacteria. Annu Rev 14. Batista AP, Kletzin A, Pereira MM (2008) The dihydrolipoamide dehydrogenase from Microbiol 55:21–48. the crenarchaeon Acidianus ambivalens. FEMS Microbiol Lett 281:147–154. 2. Ortenberg R, Gon S, Porat A, Beckwith J (2004) Interactions of glutaredoxins, 15. Newton GL, et al. (2009) Bacillithiol is an antioxidant thiol produced in Bacilli. Nat ribonucleotide reductase, and components of the DNA replication system of Chem Biol 5:625–627. Escherichia coli. Proc Natl Acad Sci USA 101:7439–7444. 16. Newton GL, et al. (1996) Distribution of thiols in microorganisms: Mycothiol is a major 3. Gon S, et al. (2006) A novel regulatory mechanism couples deoxyribonucleotide thiol in most actinomycetes. J Bacteriol 178:1990–1995. synthesis and DNA replication in Escherichia coli. EMBO J 25:1137–1147. 17. Håkansson AP, Smith AW (2007) Enzymatic characterization of dihydrolipoamide 4. Yamamoto Y, et al. (2008) Mutant AhpC peroxiredoxins suppress thiol-disulfide redox dehydrogenase from Streptococcus pneumoniae harboring its own substrate. J Biol deficiencies and acquire deglutathionylating activity. Mol Cell 29:36–45. Chem 282:29521–29530. 5. Faulkner MJ, Veeravalli K, Gon S, Georgiou G, Beckwith J (2008) Functional plasticity 18. Bringas R, Fernandez J (1995) A lipoamide dehydrogenase from Neisseria meningitidis of a peroxidase allows evolution of diverse disulfide-reducing pathways. Proc Natl has a lipoyl domain. Proteins 21:303–306. Acad Sci USA 105:6735–6740. 19. Krüger N, Oppermann FB, Lorenzl H, Steinbüchel A (1994) Biochemical and molecular 6. Mattevi A, Obmolova G, Sokatch JR, Betzel C, Hol WG (1992) The refined crystal characterization of the Clostridium magnum acetoin dehydrogenase enzyme system. structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at J Bacteriol 176:3614–3630. 2.45 A resolution. Proteins 13:336–351. 20. Smith AW, Roche H, Trombe MC, Briles DE, Håkansson A (2002) Characterization of 7. Guest JR, Creaghan IT (1972) Lipoamide dehydrogenase mutants of Escherichia coli K the dihydrolipoamide dehydrogenase from Streptococcus pneumoniae and its role in 12. Biochem J 130:8P. pneumococcal infection. Mol Microbiol 44:431–448. 8. Morris TW, Reed KE, Cronan JE, Jr. (1995) Lipoic acid metabolism in Escherichia coli: 21. Wakeham MC, Jones MR (2005) Rewiring photosynthesis: Engineering wrong-way The lplA and lipB genes define redundant pathways for ligation of lipoyl groups to electron transfer in the purple bacterial reaction centre. Biochem Soc Trans 33: apoprotein. J Bacteriol 177:1–10. 851–857. 9. Vlamis-Gardikas A (2008) The multiple functions of the thiol-based electron flow 22. Noirel J, Ow SY, Sanguinetti G, Wright PC (2009) Systems biology meets synthetic pathways of Escherichia coli: Eternal concepts revisited. Biochimica et Biophysica Acta biology: A case study of the metabolic effects of synthetic rewiring. Mol Biosyst 5: 1780:1170–1200. 1214–1223. 10. Porras P, et al. (2002) Glutaredoxins catalyze the reduction of glutathione by 23. Goodarzi H, et al. (2010) Regulatory and metabolic rewiring during laboratory dihydrolipoamide with high efficiency. Biochem Biophys Res Commun 295: evolution of ethanol tolerance in E. coli. Mol Syst Biol 6:378. 1046–1051. 24. Bryk R, Lima CD, Erdjument-Bromage H, Tempst P, Nathan C (2002) Metabolic 11. Stewart EJ, Aslund F, Beckwith J (1998) Disulfide bond formation in the Escherichia enzymes of mycobacteria linked to antioxidant defense by a thioredoxin-like protein. coli cytoplasm: An in vivo role reversal for the thioredoxins. EMBO J 17:5543–5550. Science 295:1073–1077. 12. Bessette PH, Aslund F, Beckwith J, Georgiou G (1999) Efficient folding of proteins with 25. Cussiol JRR, Alegria TGP, Szweda LI, Netto LES (2010) Ohr (organic hydroperoxide multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci USA 96: resistance protein) possesses a previously undescribed activity, lipoyl-dependent 13703–13708. peroxidase. J Biol Chem 285:21943–21950. 13. Beeby M, et al. (2005) The genomics of disulfide bonding and protein stabilization in 26. Guzman L-M, Barondess JJ, Beckwith J (1992) FtsL, an essential cytoplasmic membrane thermophiles. PLoS Biol 3:e309. protein involved in cell division in Escherichia coli. J Bacteriol 174:7716–7728.

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