Miller JR, Busby RW, Jordan SW, Cheek J, Henshaw TF, Ashley GW, Broderick JB, Cronan JE Jr, Marletta MA
Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein
▸ Abstract
The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been observed. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein containing a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry.
Biochemistry
2000;39(49):15166-15178
| PubMed ID:
11106496
Zhao X, Miller JR, Jiang Y, Marletta MA, Cronan JE
Assembly of the covalent linkage between lipoic acid and its cognate enzymes
▸ Abstract
Lipoic acid is synthesized from octanoic acid by insertion of sulfur atoms at carbons 6 and 8 and is covalently attached to a pyruvate dehydrogenase (PDH) subunit. We show that sulfur atoms can be inserted into octanoyl moieties attached to a PDH subunit or a derived domain. Escherichia coli lipB mutants grew well when supplemented with octanoate in place of lipoate. Octanoate growth required both lipoate protein ligase (LplA) and LipA, the sulfur insertion protein, suggesting that LplA attached octanoate to the dehydrogenase and LipA then converted the octanoate to lipoate. This pathway was tested by labeling a PDH domain with deuterated octanoate in an E. coli strain devoid of LipA activity. The labeled octanoyl domain was converted to lipoylated domain upon restoration of LipA. Moreover, octanoyl domain and octanoyl-PDH were substrates for sulfur insertion in vitro.
Chem Biol.
2003;10(12):1293-1302
| PubMed ID:
14700636
Cicchillo RM, Iwig DF, Jones AD, Nesbitt NM, Baleanu-Gogonea C, Souder MG, Tu L, Booker SJ
Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid
▸ Abstract
Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various alpha-keto acids, as well as the cleavage of glycine into CO(2) and NH(3), with concomitant transfer of its alpha-carbon to tetrahydrofolate, generating N(5),N(10)-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochemical studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-l-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA(*)), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming l-methionine in addition to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated derivative of the lipoyl-bearing subunit (E(2)) of the pyruvate dehydrogenase complex [Zhao, S., Miller, J. R., Jian, Y., Marletta, M. A., and Cronan, J. E., Jr. (2003) Chem. Biol. 10, 1293-1302]. Herein, we show that the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, we show that the 5'-dA(*) acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d(15)]H-protein to 5'-deoxyadenosine. Last, our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.
Biochemistry
2004;43(21):6378-6386
| PubMed ID:
15157071
Cicchillo RM, Lee KH, Baleanu-Gogonea C, Nesbitt NM, Krebs C, Booker SJ.
Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide.
▸ Abstract
Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-l-methionine (SAM) as the precursor to a high-energy 5'-deoxyadenosyl 5'-radical (5'-dA(*)). In the LS reaction, the 5'-dA(*) is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d(15) H-protein of the glycine cleavage system-one of the substrates for LS-has been reported [Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Biochemistry 43, 6378-6386]. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X(3)-C-X(2)-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X(4)-C-X(5)-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 +/- 0.5 irons and 6.4 +/- 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5'-deoxyadenosine (5'-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 +/- 0.1 irons and 3.6 +/- 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 +/- 0.1 irons and 4.7 +/- 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5'-dA or the lipoyl group. Mössbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all associated iron is in the configuration [4Fe-4S](2+). When wild-type LS was reconstituted with (57)Fe and sodium sulfide, it harbored considerably more iron (13.8 +/- 0.6) and sulfide (13.1 +/- 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5'-dA and 0.36 equiv of the lipoyl group. Mössbauer spectroscopy of this protein revealed that only approximately 67% +/- 6% of the iron is in the form of [4Fe-4S](2+) clusters, amounting to 9.2 +/- 0.4 irons and 8.8 +/- 0.1 sulfides or 2 [4Fe-4S](2+) clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the Mössbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs.
Biochemistry
2004;43(37):11770-11781
| PubMed ID:
15362861
Cicchillo RM, Booker SJ.
Mechanistic investigations of lipoic acid biosynthesis in Escherichia coli: both sulfur atoms in lipoic acid are contributed by the same lipoyl synthase polypeptide.
▸ Abstract
Lipoyl synthase catalyzes the final step in the de novo biosynthesis of the lipoyl cofactor, which is the insertion of two sulfur atoms into an octanoyl chain that is bound in an amide linkage to a conserved lysine on a lipoyl-accepting protein. We show herein that the sulfur atoms in the lipoyl cofactor are derived from lipoyl synthase itself, and that each lipoyl synthase polypeptide contributes both of the sulfur atoms to the intact cofactor.
J Am Chem Soc.
2005;127(9):2860-2861
| PubMed ID:
15740115
Booker SJ
Unraveling the pathway of lipoic acid biosynthesis
▸ Abstract
Lipoic acid is almost universally required for aerobic metabolism. However, the mechanism for its synthesis and incorporation into proteins has remained elusive. A groundbreaking study published in the December issue of Chemistry & Biology uncovers critical features of the lipoic acid biosynthetic pathway.
Comment on
Assembly of the covalent linkage between lipoic acid and its cognate enzymes. [Chem Biol. 2003]
Chem Biol.
2004;11(1):10-12
| PubMed ID:
15112987
Lanz ND, Pandelia ME, Kakar ES, Lee KH, Krebs C, Booker SJ
Evidence for a Catalytically and Kinetically Competent Enzyme-Substrate Cross-linked Intermediate in Catalysis by Lipoyl Synthase
▸ Abstract
Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis, the insertion of two sulfur atoms at C6 and C8 of an (N6-octanoyl)-lysyl residue on a lipoyl carrier protein (LCP). LS is a member of the radical SAM superfamily, enzymes that use a [4Fe-4S] cluster to effect the reductive cleavage of S-adenosyl-L-methionine (SAM) to L-methionine and a 5'-deoxyadenosyl 5'-radical (5'-dA•). In the LS reaction, two equivalents of the 5'-dA• are generated sequentially to abstract hydrogen atoms from C6 and C8 of the appended octanoyl group, initiating sulfur insertion at these positions. The second [4Fe-4S] cluster on LS, termed the auxiliary cluster, is proposed to be the source of the inserted sulfur atoms. Herein, we provide evidence for the formation of a covalent cross-link between LS and an LCP or synthetic peptide substrate in reactions in which insertion of the second sulfur atom is slowed down significantly by deuterium substitution at C8 or by inclusion of limiting concentrations of SAM. The observation that the proteins elute simultaneously by anion-exchange chromatography but are separated by aerobic sodium dodecyl sulfate-polyacrylamide gel-electrophoresis is consistent with their linkage through the auxiliary cluster that is sacrificed during turnover. Mössbauer spectroscopy confirms that one of the two [4Fe-4S] clusters of LS -presumably the auxiliary cluster - is modified or degraded as a function of extent of turnover. Generation of the cross-link species with a small, unlabeled (N6-octanoyl)-lysyl-containing peptide substrate allowed demonstration of both its chemical and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction.
Biochemistry
2014;53(28):4557-4572
| PubMed ID:
24901788
Cronan JE
The structure of lipoyl synthase, a remarkable enzyme that performs the last step of an extraordinary biosynthetic pathway
▸ Abstract
Lipoic acid is assembled on its cognate proteins (e.g. the E2 subunit of pyruvate dehydrogenase). An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthetase to a specific lysine residue of the cognate protein followed by sulfur insertion at C6 and C8 of the octanoyl chain. The challenging chemistry of this last step is performed by the radical S-adenosylmethionine (SAM) enzyme lipoyl synthase (LipA). In this issue of the Biochemical Journal, Harmer et al. report the first crystal structure of a lipoyl synthase and demonstrate that it contains two [4Fe-4S] clusters, the canonical radical SAM cluster plus a second auxiliary cluster having an unprecedented serine ligand. The structure provides strong support for the model in which the auxiliary cluster donates the lipoate sulfur atoms.
Biochem J
2014;464(1):None-None
| PubMed ID:
25341020
