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Destruction and reformation of an iron-sulfur cluster during catalysis by lipoyl synthase

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Science  20 Oct 2017:
Vol. 358, Issue 6361, pp. 373-377
DOI: 10.1126/science.aan4574

Refueling an enzyme

Lipoic acid is an eight-carbon fatty acid in which sulfur groups are appended on two carbon atoms by the enzyme lipoyl synthase (LipA). LipA provides the sulfurs from an auxiliary [4Fe-4S] cluster. McCarthy and Booker show that in Escherichia coli, the auxiliary LipA cluster is reconstituted by the iron-sulfur cluster carrier protein NfuA (see the Perspective by Rosenzweig). This occurs fast enough that LipA can act catalytically in the final step of lipoic acid biosynthesis.

Science, this issue p. 373; see also p. 307

Abstract

Lipoyl synthase (LipA) catalyzes the last step in the biosynthesis of the lipoyl cofactor, which is the attachment of two sulfhydryl groups to C6 and C8 of a pendant octanoyl chain. The appended sulfur atoms derive from an auxiliary [4Fe-4S] cluster on the protein that is degraded during turnover, limiting LipA to one turnover in vitro. We found that the Escherichia coli iron-sulfur (Fe-S) cluster carrier protein NfuA efficiently reconstitutes the auxiliary cluster during LipA catalysis in a step that is not rate-limiting. We also found evidence for a second pathway for cluster regeneration involving the E. coli protein IscU. These results show that enzymes that degrade their Fe-S clusters as a sulfur source can nonetheless act catalytically. Our results also explain why patients with NFU1 gene deletions exhibit phenotypes that are indicative of lipoyl cofactor deficiencies.

Lipoic acid is an eight-carbon, straight-chain fatty acid containing sulfhydryl groups at C6 and C8, which undergo reversible disulfide-bond formation to generate a dithiolane ring (1, 2). It is used as a redox-active cofactor in several multienzyme complexes that are involved in the oxidative decarboxylation of various α-keto acids and glycine, as well as in the oxidative degradation of acetoin (35). In these complexes, lipoic acid is attached through an amide linkage to a conserved lysyl residue of a lipoyl carrier protein (LCP), producing a 14 Å “swinging arm” that allows its dithiolane ring to access multiple active sites. A well-known role is in the pyruvate dehydrogenase complex (PDC), where it plays a central function in the conversion of pyruvate to acetyl–coenzyme A (6, 7). Other complexes in which it functions in a similar capacity are the α-ketoglutarate dehydrogenase complex (KDC), the branched-chain α-ketoacid dehydrogenase complex, the acetoin dehydrogenase complex, and the glycine cleavage system (35, 8). Deficiencies in the biosynthesis of the lipoyl cofactor or in any of the multienzyme complexes that require it result in a number of diseases, one of which is multiple mitochondrial dysfunctions syndrome, which leads to severe developmental delays, seizures, and death (9). In mammals, the complete inability to synthesize lipoic acid is embryonic lethal (10).

Although the lipoyl cofactor is structurally simple, its biosynthesis has been enigmatic. Seminal studies from the Cronan laboratory identified the pathway by which the lipoyl cofactor is biosynthesized de novo in E. coli and highlighted the roles of two dedicated proteins: octanoyltransferase (LipB), which catalyzes the transfer of an n-octanoyl moiety from octanoyl–acyl carrier protein to a conserved lysyl residue on an LCP; and lipoyl synthase (LipA; LIAS in humans and Lip5 in yeast), which catalyzes the subsequent attachment of two sulfur atoms at C6 and C8 of the aliphatic chain (1114). This pathway is conserved in all organisms that synthesize the lipoyl cofactor de novo, although slight variations have been noted (11, 1517).

LipA is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes; as such, it uses a 5′-deoxyadenosyl 5′-radical (5′-dA•) that results from the reductive cleavage of SAM to abstract hydrogen atoms (H•)—first from C6 and then from C8—to activate the aliphatic chain for sulfur attachment (1821). All radical SAM (RS) proteins contain one [4Fe-4S] cluster that supplies the electron during the reductive cleavage of SAM (22, 23). However, lipoyl synthases contain a second [4Fe-4S] cluster that has been hypothesized to be the source of the attached sulfur atoms (20, 24, 25). Consistent with this hypothesis, LipA typically catalyzes no more than one turnover during in vitro reactions because of the obligate destruction of the iron-sulfur (Fe-S) cluster (19, 21, 26). The current working hypothesis for turnover by LipA is shown in Fig. 1 (19, 20, 27). SAM binds in contact with the RS [4Fe-4S] cluster—termed [4Fe-4S]RS—and undergoes reductive cleavage to generate a 5′-dA•, which abstracts an H• from C6 of the octanoyllysyl group of an LCP. The resulting C6 radical attacks a bridging μ-sulfido ion of the auxiliary cluster, with concomitant reduction of one of the Fe3+ ions of the cluster to Fe2+ and loss of an Fe2+ ion, to afford a [3Fe-3S-1(6S)-thio-octanoyl-LCP] cluster (19, 20). A second 5′-dA•, generated from the reductive cleavage of a second SAM molecule, abstracts a C8 H•, with subsequent attack of the resulting C8 radical onto a second bridging μ-sulfido ion of the auxiliary cluster. This step results in attachment of the second sulfur atom and concomitant full or partial destruction of the cluster upon protonation of the two sulfur atoms in the nascent lipoyl group.

Fig. 1 Catalysis by LipA.

LipA contains two [4Fe-4S] clusters, one of which (the auxiliary cluster shown) is sacrificed during turnover. Catalysis proceeds by reductive cleavage of SAM to render a 5′-deoxyadenosyl 5′-radical (5′-dA•), which abstracts the C6 pro-R hydrogen atom of a pendant n-octanoamide chain residing on a lipoyl carrier protein (H protein of the glycine cleavage system shown). The resulting C6 substrate radical attacks one of the sulfide ions of the auxiliary cluster, which is followed by loss of an Fe2+ ion to afford a [3Fe-3S-1(6S)-thio-octanoamide]H protein intermediate (labeled as monothiolated octanoyl H protein). A second reductive cleavage of SAM generates a second 5′-dA•, which abstracts an H• from C8 of the thio-octanoamide H protein intermediate. The resulting C8 substrate radical attacks a second sulfide ion of the auxiliary cluster, which is followed by the addition of two protons and the loss of three Fe2+ ions and two S2– ions to generate the lipoyl group in its reduced form.

If the current proposed mechanism for LipA catalysis reflects the in vivo mechanism, LipA would be a substrate rather than an enzyme, given that it is irreversibly consumed in the reaction. Hence, it is likely that there is a system responsible for either the repair of a partially degraded cluster or the insertion of a newly assembled [4Fe-4S] cluster into the LipA active site. Although Fe-S cluster assembly can occur spontaneously in vitro, it has been established that the in vivo process involves a complex network of proteins that is highly regulated (2833). Herein, we provide evidence that E. coli NfuA, an Fe-S cluster–containing protein that has been suggested to serve as an intermediate in Fe-S cluster delivery, confers catalytic properties on E. coli LipA.

A number of studies have provided strong evidence that NFU1 and BOLA3 are involved in lipoic acid production in mammalian and yeast cells. For example, mutations in either NFU1 or BOLA3, previously considered to encode alternative scaffold proteins in Fe-S cluster biosynthesis, were found to cause a fatal deficiency of multiple respiratory chain and 2-oxoacid dehydrogenase enzymes. These deficiencies were thought to be due to a lack of the lipoyl group on the LCPs of the PDC and KDC, as well as defects in complexes I and II of the mitochondrial respiratory chain (34, 35). More recent studies directly linked NFU1 and BOLA3 to lipoic acid biosynthesis in mammalian cells and yeast, and it was determined that the proteins participated at some late stage in the process (36).

To assess the effect of NfuA—a bacterial homolog of NFU1—on the LipA reaction, we carried out molecular sieve chromatography (MSC) to first establish whether NfuA associates with LipA. E. coli NfuA was overproduced with an N-terminal hexahistidine (His6) tag to allow purification by immobilized metal affinity chromatography (fig. S1). The purified protein contained 1.8 ± 0.2 sulfide and 2.3 ± 0.2 iron ions per polypeptide; however, its ultraviolet-visible (UV-vis) spectrum suggested the presence of [4Fe-4S] clusters (Fig. 2A). Moreover, reconstitution of as-isolated NfuA with additional iron and sulfide did not lead to more cluster incorporation. This stoichiometry is consistent with the presence of one [4Fe-4S] cluster per dimer of polypeptides. LipA alone (Fig. 2B, dotted trace) eluted at 59.6 ml by MSC, exhibiting an experimentally calculated mass of 44.1 kDa (theoretical mass, 38.2 kDa) based on the elution profiles of a suite of standards (Fig. 2B and fig. S2). NfuA alone (dashed trace) eluted at 64.6 ml, exhibiting an experimentally calculated mass of 28.9 kDa (theoretical mass, 25.6 kDa). The sample containing both LipA and NfuA (solid trace) showed an elution at 54.6 ml, corresponding to an experimentally calculated mass of 67.2 kDa, which suggested a 1:1 heterodimer of LipA and NfuA (theoretical mass, 63.8 kDa). To confirm the results obtained by MSC, we subjected fractions from the two peaks observed in the LipA + NfuA trace to SDS–polyacrylamide gel electrophoresis (PAGE). As shown in Fig. 2C, the major peak (lane 2) contained both NfuA and LipA. In these experiments, NfuA migrates as a monomer and can interact with LipA as a monomer. In previous similar characterizations of NfuA, it was stated to migrate as a homodimer by MSC; however, the experimental methods and data were not provided in detail (37, 38). The UV-vis spectrum of NfuA alone upon elution from the column reveals that it had lost its cluster (fig. S3). By contrast, LipA eluted from the column with both of its clusters intact. However, when NfuA was chromatographed at higher concentrations, as during its purification, it eluted with its [4Fe-4S] cluster intact. These observations are consistent with a comparatively less stable cluster on NfuA that is required for dimerization, as has been suggested previously (3739).

Fig. 2 LipA is catalytic in the presence of NfuA.

(A) UV-vis spectrum of 15 μM as-isolated E. coli NfuA. (B) Interaction between LipA and NfuA monitored by molecular sieve chromatography. Dashed line, 100 μM NfuA alone; NfuA elutes (64.6 ml) with an experimentally calculated molecular mass of 28.9 kDa (theoretical mass, 25.6 kDa). Dotted line, 100 μM LipA alone; LipA elutes (59.6 ml) with an experimentally calculated molecular mass of 44.1 kDa (theoretical mass, 38.2 kDa). Solid line, 100 μM LipA + 100 μM NfuA; a complex between LipA and NfuA elutes (54.6 ml) with an experimentally calculated molecular mass of 67.2 kDa (theoretical mass, 63.8 kDa). (C) SDS-PAGE gel of fraction represented by solid line in (B) (lane 2). Lane 1, molecular mass markers; lane 3, NfuA standard. (D) Addition of NfuA into a LipA reaction gives rise to additional turnovers. Triangles, 6(S)-thio-octanoyllysyl intermediate; squares, lipoyl product. The reaction contained 50 μM LipA, 600 μM peptide substrate analog [Glu-Ser-Val-(N6-octanoyl)Lys-Ala-Ala-Ser-Asp], 0.5 μM SAH nucleosidase, 2 mM dithionite, and 1 mM SAM. Reactions were conducted at room temperature; at the time indicated by the arrow, 200 μM NfuA was added to the reaction. (E) Inclusion of NfuA (200 μM) in LipA reaction mixtures gives rise to multiple turnovers. Squares, lipoyl product in the absence of NfuA; triangles, lipoyl product in the presence of NfuA. The reaction conditions were as described in (D). (F) The LipA reaction under catalytic conditions. The reaction conditions were as described in (A), except that the concentration of LipA was 10 μM (7 μM active) and the concentration of NfuA was 400 μM.

Shown in Fig. 2D is a reaction depicting LipA turnover using an 8–amino acid peptide substrate analog containing the octanoyllysyl residue [Glu-Ser-Val-(N6-octanoyl)Lys-Ala-Ala-Ser-Asp] as it undergoes modification (19). The triangles show formation and decay of the monothiol-containing intermediate; the squares show formation of the lipoylated peptide product. As is typical, formation of the product leveled off around the concentration of the enzyme (50 μM). When NfuA (200 μM polypeptide; 100 μM [4Fe-4S] cluster) was injected into the reaction after a single turnover by LipA (150 min), rapid formation of additional lipoyl-containing product and monothiol-containing intermediate was observed. A similar result is shown in Fig. 2E, wherein NfuA was added at the beginning of the reaction. In this instance, more than two additional turnovers (triangles) took place above that observed when NfuA was omitted (squares). Moreover, when the concentration of LipA in the reaction was lowered (7 μM) as the concentration of NfuA was raised (400 μM), the reaction became catalytic (Fig. 2F). The absence of an initial burst of 1 equivalent of product that is followed by a slow phase indicates that cluster transfer from NfuA to LipA is not rate-limiting during catalysis.

The model for LipA catalysis shown in Fig. 1 predicts that upon one full turnover, two sulfide ions from the auxiliary [4Fe-4S] cluster are incorporated into the lipoyl product, while the remaining two sulfide ions are released into solution along with four ferrous ions. To assess this stoichiometry more rigorously, we conducted reactions with NfuA that was overproduced in the absence of its Fe-S cluster and then reconstituted with iron and 34S-labeled sulfide. Shown in Fig. 3A is a reaction with 20 μM LipA containing Fe-S clusters composed of sulfide at natural abundance (~95% 32S) and 100 μM NfuA containing Fe-S clusters composed of 34S-labeled (~99%) sulfide. An initial burst of lipoyl product containing two 32S sulfurs was observed (circles), which is consistent with previous results that indicate that the auxiliary cluster of LipA is used as the source of inserted sulfur atoms (19, 20, 40). Surprisingly, however, the amount of lipoyl product containing two 32S atoms was more than 50% greater than that expected (33 μM versus 20 μM); this finding suggests that the enzyme can potentially direct all four sulfide ions from the cluster into the lipoyl product rather than release two of them into solution during each turnover. Lipoyl product containing two 34S atoms was also observed (triangles), confirming that the [4Fe-4S] cluster on NfuA can be used to reconstitute the auxiliary cluster on LipA. Formation of the lipoyl product containing two 34S atoms took place with a pronounced lag, as expected for a reaction in which the Fe-S cluster on LipA is consumed before NfuA transfers a labeled cluster to LipA. In this experiment, the amount of 34S-labeled lipoyl product was limited by the concentration of holo NfuA in the reaction mixture. When the experiment was conducted with excess NfuA (400 μM NfuA and ~7 μM active LipA), production of ~1.5 equivalents of the 32S-labeled lipoyl product was followed by production of multiple equivalents of the 344S-labeled lipoyl product (Fig. 3B). We also observed the slow formation of a lipoyl group containing one 32S atom and one 34S atom (Fig. 3A), which we believe derives from aberrantly released sulfide that is subsequently used to reconstitute another auxiliary cluster during the reaction. Consistent with this explanation, when reactions were conducted in the presence of 0.8 mM sodium sulfide but in the absence of NfuA, additional product formation was observed; however, this additional formation of product was not as fast or as extensive as when NfuA was present (Fig. 3C). Similarly, when IscS, cysteine, and dithiothreitol (DTT) were included in reaction mixtures, a slight increase in product was observed (Fig. 3D).

Fig. 3 Cluster transfer from NfuA to LipA is direct.

(A) Formation of lipoyl product in the presence of NfuA reconstituted with 34S-labeled sulfide. Circles, (32S,32S)–containing lipoyl product; triangles, (34S,34S)–containing lipoyl product; squares, (32S,34S)–containing lipoyl product. The reaction contained 20 μM LipA, 100 μM 34S-labeled NfuA, 600 μM peptide substrate analog, 0.5 μM SAH nucleosidase, 2 mM dithionite, and 1 mM SAM. (B) Formation of lipoyl product in the presence of NfuA reconstituted with 34S-labeled sulfide and under catalytic conditions. The reaction conditions were as described above, except that the concentration of LipA was 10 μM and the concentration of 34S-labeled NfuA was 400 μM. Circles, (32S,32S)–containing lipoyl product; triangles, (34S,34S)–containing lipoyl product. (C) Effect of extraneous iron and sulfide on the activity of LipA. Solid squares, LipA reaction (50 μM) in the absence of NfuA or iron and sulfide; open squares, LipA reaction in the presence of 0.8 mM FeCl3 and 0.8 mM Na2S; triangles, LipA reaction in the presence of 200 μM NfuA. Other reaction components were as described in (A). (D) Effect of IscS on the LipA reaction. Triangles, LipA reaction in the presence of IscS; squares, LipA reaction in the absence of IscS. Reactions were as described in (A), except that they contained 100 μM LipA, 200 μM IscS, 5 mM cysteine, 5 mM DTT, and no NfuA. (E) Effect of citrate on NfuA’s enhancement of LipA catalysis. Open squares, 100 μM LipA; solid squares, 100 μM LipA + 5 mM citrate; open circles, 100 μM LipA + 1 mM NfuA; solid circles, 100 μM LipA + 1 mM NfuA + 5 mM citrate. (F) Effect of extraneous sulfide on LipA catalysis in the presence of NfuA. Squares, 25 μM LipA + 400 μM NfuA monitoring product containing two 32S atoms; triangles, 25 μM LipA + 400 μM NfuA in the presence of 1 mM Na234S in which the lipoyllysyl product containing two 32S atoms is monitored; solid circles, 25 μM LipA + 400 μM NfuA in the presence of 1 mM Na234S monitoring the 32S/34S mixed-labeled product; open circles, 25 μM LipA + 400 μM NfuA in the presence of 1 mM Na234S monitoring the 34S/34S-labeled product.

To show that the effect of NfuA on the LipA reaction involves a direct transfer of the cluster from NfuA to LipA, rather than the release of iron and sulfide into solution followed by reconstitution of the auxiliary cluster, we conducted two additional experiments. In one experiment, the effect of NfuA (1 mM) on LipA (100 μM) catalysis was measured in the presence of 5 mM citrate, which can chelate released iron from NfuA and prevent it from being used in the reconstitution of LipA during turnover (41). The presence of citrate had no effect on NfuA’s ability to enhance LipA turnover (Fig. 3E). In a second experiment, the effect of NfuA (400 μM) on LipA (25 μM) catalysis was measured in the presence of 1 mM Na234S. If the enhanced effect of NfuA were mediated through release of its iron and sulfide into solution, substantial incorporation of 34S in the lipoyl product after the initial turnover would be expected (42). When the reaction was conducted in the presence of Na234S, the formation of lipoyl product containing two 32S atoms was similar to that when the reaction was conducted in the absence of Na234S; a very small amount of lipoyl product containing one 34S atom and one 32S atom was observed, whereas virtually no product containing two 34S atoms was observed (Fig. 3F). These results indicate that release of iron and sulfide from NfuA into solution followed by its use in reconstituting the auxiliary cluster of LipA is unlikely.

The effect of NfuA on the growth and viability of E. coli was assessed in two previous studies (37, 43). In one study, ΔnfuA strains grew as well as wild-type strains in enriched media, except under conditions of oxidative stress (paraquat administration) or iron starvation (2,2′-dipyridyl administration) (37). In a second study, a ΔnfuA strain displayed a growth curve in Luria-Bertani (LB) medium that was similar to that of the wild-type control. However, ΔnfuA strains that also carried isc operon gene deletions displayed an impaired growth rate (38). Because E. coli can incorporate exogenous lipoic acid present in rich or LB media into LCPs via a pathway that is independent of LipA, we investigated whether an E. coli ΔnfuA strain could grow in M9 minimal medium lacking lipoic acid and using glucose as a carbon source. The ΔnfuA strain did not exhibit pronounced growth defects relative to the control; moreover, neither the addition of exogenous lipoic acid nor succinate plus acetate (which bypasses the requirement for lipoic acid) had a measurable effect on the overall growth rate (Fig. 4A). By contrast, E. coli lipA mutants could not grow under these conditions. These results differ from the extreme phenotypes observed in humans or yeast for NFU1 or BOLA3 deletion strains (3436).

Fig. 4 E. coli IscU serves as a second pathway for cluster regeneration.

(A) Effect of NfuA on growth of E. coli in M9 minimal medium using glucose as a carbon source. Solid circles, wild-type BW25113; squares, BW25113:ΔnfuA; open circles, BW25113:ΔnfuA + 25 μM lipoic acid; triangles, BW25113:ΔnfuA + 5 mM succinate/5 mM acetate. (B) Effect of IscU on LipA catalysis. Squares, LipA only; triangles, LipA + IscU; circles, LipA + NfuA. (C) Effect of IscU, HscA, and HscB on LipA catalysis. Squares, LipA only; circles, LipA + IscU; triangles, LipA + IscU + HscA and HscB. (D) Effect of NfuA, HscA, and HscB on LipA catalysis. Squares, LipA only; circles, LipA + NfuA; triangles, LipA + NfuA + HscA and HscB. Reactions contained 20 μM LipA, 400 μM octanoyllysyl-containing peptide substrate, 2 mM SAM, 2 mM dithionite, 2 mM ATP, 100 mM MgCl2, and 0.5 μM SAH nucleosidase. When appropriate, IscU, HscA, and HscB were each added to a final concentration of 200 μM. (E) Effect of extraneous sulfide on LipA catalysis in the presence of IscU. Circles, 25 μM LipA-only control; squares, 25 μM LipA and 400 μM IscU in the presence of 1 mM Na234S in which the lipoyl product containing two 32S atoms is monitored; solid triangles, 25 μM LipA + 400 μM IscU in the presence of 1 mM Na234S monitoring the 32S/34S mixed-labeled product; open triangles, 25 μM LipA + 400 μM IscU in the presence of 1 mM Na234S monitoring the the 34S/34S double-labeled product.

The observation that E. coli ΔnfuA strains do not exhibit severe growth defects in the absence of lipoic acid suggests that a second pathway exists for the regeneration of LipA’s auxiliary cluster in E. coli. Given that an exacerbated effect was previously observed in an ΔnfuA:ΔiscU strain relative to strains harboring single deletions of the two genes, we assessed whether E. coli IscU could render LipA catalytic in the absence of NfuA (37). E. coli IscU was overproduced and isolated in its apo form, and its [4Fe-4S] cluster was then reconstituted using previously established methods (44). Indeed, when E. coli IscU containing a [4Fe-4S] cluster was included in excess of LipA, additional turnover was observed (Fig. 4B). The cochaperones HscA and HscB, encoded within the isc gene operon, function in facilitating cluster transfer from E. coli IscU to its recipient proteins, including NfuA (37, 45). However, no increase in rate or product formation was observed when HscA, HscB, MgCl2, and adenosine triphosphate (ATP) were included in the reaction with holo IscU or holo NfuA; in fact, the inclusion of HscA and HscB was slightly inhibitory (Fig. 4, C and D).

We also carried out a similar scrambling study of the effect of IscU on LipA catalysis. When the reaction was conducted with unlabeled IscU and unlabeled LipA in the presence of 1 mM Na234S, almost all of the resulting lipoyl product contained two 32S atoms; very little contained the mixed 32S/34S or the 34S/34S product (Fig. 4E). The effect of citrate on the enhancement of the LipA reaction by IscU could not be studied because IscU’s cluster was unstable under those conditions (fig. S4).

The resistance to the idea that an Fe-S cluster can act as a sulfur source during the radical-mediated sulfhydrylation of unactivated carbon centers is largely due to the consequence of the enzyme inactivating itself after only one turnover. Our finding that E. coli NfuA or IscU can reinstall the Fe-S cluster in E. coli LipA after each turnover, in a process that is not rate-limiting, suggests that this concern is no longer warranted. Our studies also most likely explain why patients with defects in NFU1, the mammalian ortholog of NfuA, display phenotypes that are consistent with lipoic acid deficiency.

Supplementary Materials

www.sciencemag.org/content/358/6361/373/suppl/DC1

Materials and Methods

Figs. S1 to S4

Tables S1 and S2

References (46, 47)

References and Notes

Acknowledgments: We thank B. Wang for assistance with the synthesis of 34S-labeled sulfide, and P. Babitzke (Penn State) for E. coli BW25113. Supported by NIH grant GM122595 and NSF grant MCB-1158486 (S.J.B.). S.J.B. is an Investigator of the Howard Hughes Medical Institute.
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