Publications

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

  McCarthy E.L., Booker S.J.

  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, ... >> More

  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 <i>Escherichia coli</i> 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 <i>E. coli</i> 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 <i>NFU1</i> gene deletions exhibit phenotypes that are indicative of lipoyl cofactor deficiencies. << Less

  Science 358:373-377(2017) [PubMed] [EuropePMC]

• Lipoic acid metabolism in Escherichia coli: isolation of null mutants defective in lipoic acid biosynthesis, molecular cloning and characterization of the E. coli lip locus, and identification of the lipoylated protein of the glycine cleavage system.

  Vanden Boom T.J., Reed K.E., Cronan J.E. Jr.

  We report the isolation and genetic characterization of novel Tn10dTc and Tn1000dKn insertion mutations in and near the lip locus of the Escherichia coli chromosome. The Tn10dTc and Tn1000dKn mutations define two genes, lipA and lipB, involved in lipoic acid biosynthesis. Two representative allele ... >> More

  We report the isolation and genetic characterization of novel Tn10dTc and Tn1000dKn insertion mutations in and near the lip locus of the Escherichia coli chromosome. The Tn10dTc and Tn1000dKn mutations define two genes, lipA and lipB, involved in lipoic acid biosynthesis. Two representative alleles (lip-2 and lip-9) from the previously reported genetic class of lipoic acid auxotrophic mutants (A. A. Herbert and J. R. Guest, J. Gen. Microbiol. 53:363-381, 1968) were assigned to the lipA complementation group. We have cloned the E. coli lip locus and developed a recombinant plasmid-based genetic system for fine-structure physical-genetic mapping of mutations in this region of the E. coli chromosome. We also report that a recombinant plasmid containing a 5.2-kbp PvuII restriction fragment from the E. coli lip locus produced three proteins of approximately 8, 12, and 36 kDa by using either a maxicell or in vitro transcription translation expression system. The 36-kDa protein was identified as the gene product encoded by the lipA locus. Finally, we have identified a previously unreported lipoylated protein that functions in the glycine cleavage system of E. coli. << Less

  J Bacteriol 173:6411-6420(1991) [PubMed] [EuropePMC]

• Self-sacrifice in radical S-adenosylmethionine proteins.

  Booker S.J., Cicchillo R.M., Grove T.L.

  The radical SAM superfamily of metalloproteins catalyze the reductive cleavage of S-adenosyl-l-methionine to generate a 5'-deoxyadenosyl radical (5'-dA*) intermediate that is obligate for turnover. The 5'-dA* acts as a potent oxidant, initiating turnover by abstracting a hydrogen atom from an appr ... >> More

  The radical SAM superfamily of metalloproteins catalyze the reductive cleavage of S-adenosyl-l-methionine to generate a 5'-deoxyadenosyl radical (5'-dA*) intermediate that is obligate for turnover. The 5'-dA* acts as a potent oxidant, initiating turnover by abstracting a hydrogen atom from an appropriate substrate. A special class of these enzymes use this strategy to functionalize unactivated C-H bonds by insertion of sulfur atoms. This review will describe the characterization of three members of this class - biotin synthase, lipoyl synthase, and MiaB protein - each of which has been shown to cannibalize itself during turnover. << Less

  Curr Opin Chem Biol 11:543-552(2007) [PubMed] [EuropePMC]

• A new metabolic link. The acyl carrier protein of lipid synthesis donates lipoic acid to the pyruvate dehydrogenase complex in Escherichia coli and mitochondria.

  Jordan S.W., Cronan J.E. Jr.

  Lipoic acid is an essential enzyme cofactor that requires covalent attachment to its cognate proteins to confer biological activity. The major lipoylated proteins are highly conserved enzymes of central metabolism, the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The classical lipoate ... >> More

  Lipoic acid is an essential enzyme cofactor that requires covalent attachment to its cognate proteins to confer biological activity. The major lipoylated proteins are highly conserved enzymes of central metabolism, the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The classical lipoate ligase uses ATP to activate the lipoate carboxyl group followed by attachment of the cofactor to a specific subunit of each dehydrogenase complex, and it was assumed that all lipoate attachment proceeded by this mechanism. However, our previous work indicated that Escherichia coli could form lipoylated proteins in the absence of detectable ATP-dependent ligase activity raising the possibility of a class of enzyme that attaches lipoate to the dehydrogenase complexes by a different mechanism. We now report that E. coli and mitochondria contain lipoate transferases that use lipoyl-acyl carrier protein as the lipoate donor. This finding demonstrates a direct link between fatty acid synthesis and lipoate attachment and also provides the first direct demonstration of a role for the enigmatic acyl carrier proteins of mitochondria. << Less

  J Biol Chem 272:17903-17906(1997) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Assembly of the covalent linkage between lipoic acid and its cognate enzymes.

  Zhao X., Miller J.R., Jiang Y., Marletta M.A., Cronan J.E. Jr.

  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 ... >> More

  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. << Less

  Chem. Biol. 10:1293-1302(2003) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Mechanistic investigations of lipoic acid biosynthesis in Escherichia coli: both sulfur atoms in lipoic acid are contributed by the same lipoyl synthase polypeptide.

  Cicchillo R.M., Booker S.J.

  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 c ... >> More

  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. << Less

  J. Am. Chem. Soc. 127:2860-2861(2005) [PubMed] [EuropePMC]

• Swinging arms and swinging domains in multifunctional enzymes: catalytic machines for multistep reactions.

  Perham R.N.

  Multistep chemical reactions are increasingly seen as important in a growing number of complex biotransformations. Covalently attached prosthetic groups or swinging arms, and their associated protein domains, are essential to the mechanisms of active-site coupling and substrate channeling in a num ... >> More

  Multistep chemical reactions are increasingly seen as important in a growing number of complex biotransformations. Covalently attached prosthetic groups or swinging arms, and their associated protein domains, are essential to the mechanisms of active-site coupling and substrate channeling in a number of the multifunctional enzyme systems responsible. The protein domains, for which the posttranslational machinery in the cell is highly specific, are crucially important, contributing to the processes of molecular recognition that define and protect the substrates and the catalytic intermediates. The domains have novel folds and move by virtue of conformationally flexible linker regions that tether them to other components of their respective multienzyme complexes. Structural and mechanistic imperatives are becoming apparent as the assembly pathways and the coupling of multistep reactions catalyzed by these dauntingly complex molecular machines are unraveled. << Less

  Annu Rev Biochem 69:961-1004(2000) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein.

  Miller J.R., Busby R.W., Jordan S.W., Cheek J., Henshaw T.F., Ashley G.W., Broderick J.B., Cronan J.E. Jr., Marletta M.A.

  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 ... >> More

  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. << Less

  Biochemistry 39:15166-15178(2000) [PubMed] [EuropePMC]

• Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid.

  Cicchillo R.M., Iwig D.F., Jones A.D., Nesbitt N.M., Baleanu-Gogonea C., Souder M.G., Tu L., Booker S.J.

  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 tetrahy ... >> More

  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. << Less

  Biochemistry 43:6378-6386(2004) [PubMed] [EuropePMC]