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MetaCyc Enzyme: lipoyl synthase

Gene: lipA Accession Numbers: EG11306 (MetaCyc), b0628, ECK0621

Synonyms: lip

Species: Escherichia coli K-12 substr. MG1655

Subunit composition of lipoyl synthase = [LipA]2
         lipoyl synthase = LipA

Summary:
Lipoate synthase catalyzes the final step of de novo lipoate biosynthesis, the insertion of sulfur into the octanoyl side chain of an octanoylated E2 domain to form the lipoate moiety [Reed93a, Miller00, Zhao03]. Lipoate modification of the E2 subunits is important for the function of pyruvatedeh-cplx [Herbert75, Stepp81, Reed93a], α-ketoglutarate dehydrogenase [Herbert75, Stepp81, Reed93a], and the glycine cleavage system [Vanden91, Reed93a].

Lipoate synthase is a homodimer containing iron-sulfur clusters [Ollagnierde99, Busby99a, OllagnierDe00, Miller00]. Coexpression with the isc operon increases the yield of overexpressed soluble LipA [Kriek03]. LipA belongs to the radical SAM superfamily of enzymes [Sofia01]. The enzyme uses octanoyl side chains (but not free octanoate) as substrate [Miller00, Zhao03]. The 5'-dA· radicals, generated from SAM, act directly on the octanoyl substrate. Two equivalents of 5'-dA· sequentially abstract hydrogen atoms from C6 and C8 of the octanoyl group, preparing it for sulfur insertion [Cicchillo04]. LipA contains two distinct 4Fe-4S clusters per polypeptide which are both required for catalytic activity [Cicchillo04b], and one of which may be the source of the sulfur atoms required for synthesis of the lipoyl group [Cicchillo05a]. Evidence of a covalently linked monothiolated reaction intermediate and a partially degraded Fe-S cluster has been obtained [Lanz14].

LipA and LipB interact directly and independently with the E2 protein of both pyruvatedeh-cplx and α-ketoglutarate dehydrogenase, but not GcvH of the glycine cleavage system [Hassan11].

The lip-2 and lip-9 mutations, which cause lipoic acid auxotrophy, are lipA alleles [Vanden91]; lip-2 encodes an S307F change and lip-9 encodes an E195K change [Hayden93]. The lipoate auxotrophy of a lipA mutant is rescued by 6-thiooctanoate or 8-thiooctanoate [Reed93a]. lipA mutants have an extended lifespan [Gonidakis10].

A PdhR binding site that was predicted to be involved in regulation of lipA expression has been identified [Kaleta10], but was shown to be nonfunctional [Feng].

Reviews: [Cronan14, Booker07, Cronan05, Booker04, Frey01, Marquet01]

Citations: [Herbert68, Chang91, Creaghan78, Hayden92, Inoue02, Wan02, ChoiRhee05, Gonidakis10a]

Locations: cytosol

Map Position: [658,474 <- 659,439]

Molecular Weight of Polypeptide: 36.072 kD (from nucleotide sequence), 36 kD (experimental) [Vanden91 ]

Molecular Weight of Multimer: 84.6 kD (experimental) [Miller00]

Unification Links: ASAP:ABE-0002155 , CGSC:31534 , DIP:DIP-48008N , EchoBASE:EB1283 , EcoGene:EG11306 , EcoliWiki:b0628 , Mint:MINT-1310776 , ModBase:P60716 , OU-Microarray:b0628 , PortEco:lipA , PR:PRO_000023092 , Pride:P60716 , Protein Model Portal:P60716 , RefSeq:NP_415161 , RegulonDB:EG11306 , SMR:P60716 , String:511145.b0628 , UniProt:P60716

Relationship Links: InterPro:IN-FAMILY:IPR003698 , InterPro:IN-FAMILY:IPR006638 , InterPro:IN-FAMILY:IPR007197 , InterPro:IN-FAMILY:IPR013785 , Panther:IN-FAMILY:PTHR10949 , Pfam:IN-FAMILY:PF04055 , Smart:IN-FAMILY:SM00729

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0009107 - lipoate biosynthetic process Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, Reed93a]
GO:0009249 - protein lipoylation Inferred from experiment Inferred by computational analysis [GOA06, Cicchillo04]
GO:0055114 - oxidation-reduction process Inferred from experiment [Reed93a]
Molecular Function: GO:0003826 - alpha-ketoacid dehydrogenase activity Inferred from experiment [Reed93a]
GO:0005515 - protein binding Inferred from experiment [Hassan11]
GO:0016992 - lipoate synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, Cicchillo04]
GO:0042803 - protein homodimerization activity Inferred from experiment [Miller00]
GO:0051539 - 4 iron, 4 sulfur cluster binding Inferred from experiment Inferred by computational analysis [UniProtGOA11a, GOA06, GOA01a, Cicchillo04b, OllagnierDe00]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11a]
GO:0051536 - iron-sulfur cluster binding Inferred by computational analysis [UniProtGOA11a, GOA01a]
Cellular Component: GO:0005737 - cytoplasm Inferred from experiment Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA06, Cicchillo04]
GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]

MultiFun Terms: metabolism biosynthesis of building blocks cofactors, small molecule carriers lipoate

Credits:
Imported from EcoCyc 27-Jan-2015 by Paley S , SRI International


Enzymatic reaction of: lipoyl synthase

Synonyms: lipoic acid synthase, lipoate synthase

EC Number: 2.8.1.8

a [lipoyl-carrier protein] N6-octanoyl-L-lysine + 2 S-adenosyl-L-methionine + 2 a sulfurated [sulfur carrier] + 2 a reduced [2Fe-2S] ferredoxin <=> a [lipoyl-carrier protein] N6-lipoyl-L-lysine + 2 5'-deoxyadenosine + 2 L-methionine + 2 an unsulfurated [sulfur carrier] + 2 an oxidized [2Fe-2S] ferredoxin

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is favored in the direction shown.

In Pathways: lipoate biosynthesis and incorporation II , lipoate biosynthesis and incorporation I

Credits:
Imported from EcoCyc 27-Jan-2015 by Paley S , SRI International

Cofactors or Prosthetic Groups: a [4Fe-4S] iron-sulfur cluster [Cicchillo04b, Miller00]


Sequence Features

Feature Class Location Citations Comment
Sequence-Conflict 35
[Reed93a, Hayden92, UniProt10a]
UniProt: (in Ref. 1 and 2);
Mutagenesis-Variant 68 -> 79
[Cicchillo04b, UniProt11a]
UniProt: Loss of 1 4Fe-4S cluster binding. Loss of activity.
Metal-Binding-Site 68
[UniProt10a]
UniProt: Iron-sulfur 1 (4Fe-4S);
Metal-Binding-Site 73
[UniProt10a]
UniProt: Iron-sulfur 1 (4Fe-4S);
Metal-Binding-Site 79
[UniProt10a]
UniProt: Iron-sulfur 1 (4Fe-4S);
Mutagenesis-Variant 94 -> 101
[Cicchillo04b, UniProt11a]
UniProt: Loss of 1 4Fe-4S cluster binding. Loss of activity.
Metal-Binding-Site 94
[UniProt10a]
UniProt: Iron-sulfur 2 (4Fe-4S-S-AdoMet);
Metal-Binding-Site 98
[UniProt10a]
UniProt: Iron-sulfur 2 (4Fe-4S-S-AdoMet);
Metal-Binding-Site 101
[UniProt10a]
UniProt: Iron-sulfur 2 (4Fe-4S-S-AdoMet);

History:
10/20/97 Gene b0628 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11306; confirmed by SwissProt match.


References

Booker04: Booker SJ (2004). "Unraveling the pathway of lipoic acid biosynthesis." Chem Biol 11(1);10-2. PMID: 15112987

Booker07: Booker SJ, Cicchillo RM, Grove TL (2007). "Self-sacrifice in radical S-adenosylmethionine proteins." Curr Opin Chem Biol 11(5);543-52. PMID: 17936058

Busby99a: Busby RW, Schelvis JP M, Yu DS, Babcock GT, Marletta MA (1999). "Lipoic Acid Biosynthesis:  LipA Is an Iron−Sulfur Protein." Journal of the American Chemical Society 121(19);4706-4707.

Chang91: Chang YY, Cronan JE, Li SJ, Reed K, Vanden Boom T, Wang AY (1991). "Locations of the lip, poxB, and ilvBN genes on the physical map of Escherichia coli." J Bacteriol 173(17);5258-9. PMID: 1832150

ChoiRhee05: Choi-Rhee E, Cronan JE (2005). "A nucleosidase required for in vivo function of the S-adenosyl-L-methionine radical enzyme, biotin synthase." Chem Biol 12(5);589-93. PMID: 15911379

Cicchillo04: Cicchillo RM, Iwig DF, Jones AD, Nesbitt NM, Baleanu-Gogonea C, Souder MG, Tu L, Booker SJ (2004). "Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid." Biochemistry 43(21);6378-86. PMID: 15157071

Cicchillo04b: Cicchillo RM, Lee KH, Baleanu-Gogonea C, Nesbitt NM, Krebs C, Booker SJ (2004). "Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide." Biochemistry 43(37);11770-81. PMID: 15362861

Cicchillo05a: Cicchillo RM, Booker SJ (2005). "Mechanistic investigations of lipoic acid biosynthesis in Escherichia coli: both sulfur atoms in lipoic acid are contributed by the same lipoyl synthase polypeptide." J Am Chem Soc 127(9);2860-1. PMID: 15740115

Creaghan78: Creaghan IT, Guest JR (1978). "Succinate dehydrogenase-dependent nutritional requirement for succinate in mutants of Escherichia coli K12." J Gen Microbiol 107(1);1-13. PMID: 366070

Cronan05: Cronan JE, Zhao X, Jiang Y (2005). "Function, attachment and synthesis of lipoic acid in Escherichia coli." Adv Microb Physiol 50;103-46. PMID: 16221579

Cronan14: Cronan JE (2014). "The structure of lipoyl synthase, a remarkable enzyme that performs the last step of an extraordinary biosynthetic pathway." Biochem J 464(1);e1-3. PMID: 25341020

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Feng: Feng Y, Cronan JE "PdhR, the pyruvate dehydrogenase repressor, does not regulate lipoic acid synthesis." Res Microbiol 165(6);429-38. PMID: 24816490

Frey01: Frey PA (2001). "Radical mechanisms of enzymatic catalysis." Annu Rev Biochem 70;121-48. PMID: 11395404

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Gonidakis10: Gonidakis S, Finkel SE, Longo VD (2010). "Genome-wide screen identifies Escherichia coli TCA-cycle-related mutants with extended chronological lifespan dependent on acetate metabolism and the hypoxia-inducible transcription factor ArcA." Aging Cell 9(5);868-81. PMID: 20707865

Gonidakis10a: Gonidakis S, Finkel SE, Longo VD (2010). "E. coli hypoxia-inducible factor ArcA mediates lifespan extension in a lipoic acid synthase mutant by suppressing acetyl-CoA synthetase." Biol Chem 391(10);1139-47. PMID: 20707605

Hassan11: Hassan BH, Cronan JE (2011). "Protein-protein interactions in assembly of lipoic acid on the 2-oxoacid dehydrogenases of aerobic metabolism." J Biol Chem 286(10);8263-76. PMID: 21209092

Hayden92: Hayden MA, Huang I, Bussiere DE, Ashley GW (1992). "The biosynthesis of lipoic acid. Cloning of lip, a lipoate biosynthetic locus of Escherichia coli." J Biol Chem 267(14);9512-5. PMID: 1577793

Hayden93: Hayden MA, Huang IY, Iliopoulos G, Orozco M, Ashley GW (1993). "Biosynthesis of lipoic acid: characterization of the lipoic acid auxotrophs Escherichia coli W1485-lip2 and JRG33-lip9." Biochemistry 32(14);3778-82. PMID: 8466915

Herbert68: Herbert AA, Guest JR (1968). "Biochemical and genetic studies with lysine+methionine mutants of Escherichia coli: lipoic acid and alpha-ketoglutarate dehydrogenase-less mutants." J Gen Microbiol 53(3);363-81. PMID: 4889470

Herbert75: Herbert AA, Guest JR (1975). "Lipoic acid content of Escherichia coli and other microorganisms." Arch Microbiol 106(3);259-66. PMID: 814874

Inoue02: Inoue K, Chen J, Kato I, Inouye M (2002). "Specific growth inhibition by acetate of an Escherichia coli strain expressing Era-dE, a dominant negative Era mutant." J Mol Microbiol Biotechnol 4(4);379-88. PMID: 12125819

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Kaleta10: Kaleta C, Gohler A, Schuster S, Jahreis K, Guthke R, Nikolajewa S (2010). "Integrative inference of gene-regulatory networks in Escherichia coli using information theoretic concepts and sequence analysis." BMC Syst Biol 4;116. PMID: 20718955

Kriek03: Kriek M, Peters L, Takahashi Y, Roach PL (2003). "Effect of iron-sulfur cluster assembly proteins on the expression of Escherichia coli lipoic acid synthase." Protein Expr Purif 28(2);241-5. PMID: 12699687

Lanz14: Lanz ND, Pandelia ME, Kakar ES, Lee KH, Krebs C, Booker SJ (2014). "Evidence for a catalytically and kinetically competent enzyme-substrate cross-linked intermediate in catalysis by lipoyl synthase." Biochemistry 53(28);4557-72. PMID: 24901788

Marquet01: Marquet A, Bui BT, Florentin D (2001). "Biosynthesis of biotin and lipoic acid." Vitam Horm 61;51-101. PMID: 11153271

Miller00: Miller JR, Busby RW, Jordan SW, Cheek J, Henshaw TF, Ashley GW, Broderick JB, Cronan JE, Marletta MA (2000). "Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein." Biochemistry 39(49);15166-78. PMID: 11106496

OllagnierDe00: Ollagnier-De Choudens S, Sanakis Y, Hewitson KS, Roach P, Baldwin JE, Munck E, Fontecave M (2000). "Iron-sulfur center of biotin synthase and lipoate synthase." Biochemistry 39(14);4165-73. PMID: 10747808

Ollagnierde99: Ollagnier-de Choudens S, Fontecave M (1999). "The lipoate synthase from Escherichia coli is an iron-sulfur protein." FEBS Lett 453(1-2);25-8. PMID: 10403368

Reed93a: Reed KE, Cronan JE (1993). "Lipoic acid metabolism in Escherichia coli: sequencing and functional characterization of the lipA and lipB genes." J Bacteriol 175(5);1325-36. PMID: 8444795

Sofia01: Sofia HJ, Chen G, Hetzler BG, Reyes-Spindola JF, Miller NE (2001). "Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods." Nucleic Acids Res 29(5);1097-106. PMID: 11222759

Stepp81: Stepp LR, Bleile DM, McRorie DK, Pettit FH, Reed LJ (1981). "Use of trypsin and lipoamidase to study the role of lipoic acid moieties in the pyruvate and alpha-ketoglutarate dehydrogenase complexes of Escherichia coli." Biochemistry 20(16);4555-60. PMID: 6794598

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt11a: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

Vanden91: Vanden Boom TJ, Reed KE, Cronan JE (1991). "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." J Bacteriol 173(20);6411-20. PMID: 1655709

Wan02: Wan JT, Jarrett JT (2002). "Electron acceptor specificity of ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli." Arch Biochem Biophys 406(1);116-26. PMID: 12234497

Zhao03: Zhao X, Miller JR, Jiang Y, Marletta MA, Cronan JE (2003). "Assembly of the covalent linkage between lipoic acid and its cognate enzymes." Chem Biol 10(12);1293-302. PMID: 14700636


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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 19.0 on Tue Apr 21, 2015, biocyc13.