Escherichia coli K-12 substr. MG1655 Enzyme: lipoyl synthase

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

Synonyms: lip

Regulation Summary Diagram: ?

Regulation summary diagram for lipA

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

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 [Reed93, Miller00, Zhao03]. Lipoate modification of the E2 subunits is important for the function of pyruvate dehydrogenase [Herbert75, Stepp81, Reed93], α-ketoglutarate dehydrogenase [Herbert75, Stepp81, Reed93], and the glycine cleavage system [Vanden91, Reed93].

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 [Cicchillo04a], and one of which may be the source of the sulfur atoms required for synthesis of the lipoyl group [Cicchillo05]. 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 pyruvate dehydrogenase 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 [Reed93]. lipA mutants have an extended lifespan [Gonidakis10a].

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 [Feng14].

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

Citations: [Herbert68, Chang91, Creaghan78, Hayden92, Inoue02, Wan02, ChoiRhee05, Gonidakis10]

Locations: cytosol

Map Position: [658,474 <- 659,439] (14.19 centisomes, 51°)
Length: 966 bp / 321 aa

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

In Paralogous Gene Group: 168 (2 members)

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0009107 - lipoate biosynthetic process Inferred from experiment Inferred by computational analysis [GOA06, GOA01, Reed93]
GO:0009249 - protein lipoylation Inferred from experiment Inferred by computational analysis [GOA06, Cicchillo04]
GO:0055114 - oxidation-reduction process Inferred from experiment [Reed93]
Molecular Function: GO:0003826 - alpha-ketoacid dehydrogenase activity Inferred from experiment [Reed93]
GO:0005515 - protein binding Inferred from experiment [Hassan11]
GO:0016992 - lipoate synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, 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 [UniProtGOA11, GOA06, GOA01, Cicchillo04a, OllagnierDe00]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11]
GO:0051536 - iron-sulfur cluster binding Inferred by computational analysis [UniProtGOA11, GOA01]
Cellular Component: GO:0005737 - cytoplasm Inferred from experiment Inferred by computational analysis [UniProtGOA11a, UniProtGOA11, 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

Essentiality data for lipA knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 0.4% glucose No 37 Aerobic 7.2 0.27 No [Patrick07, Comment 3]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 4]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]

Last-Curated ? 06-Nov-2014 by Keseler I , SRI International

Enzymatic reaction of: lipoyl synthase

Synonyms: lipoic acid synthase, lipoate synthase

EC Number:

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

Cofactors or Prosthetic Groups: a [4Fe-4S] iron-sulfur cluster [Cicchillo04a, Miller00]

Sequence Features

Protein sequence of lipoyl synthase with features indicated

Feature Class Location Citations Comment
Sequence-Conflict 35
[Reed93, Hayden92, UniProt10a]
UniProt: (in Ref. 1 and 2);
Metal-Binding-Site 68
UniProt: Iron-sulfur 1 (4Fe-4S).
Mutagenesis-Variant 68 -> 79
[Cicchillo04a, UniProt11]
UniProt: Loss of 1 4Fe-4S cluster binding. Loss of activity.
Metal-Binding-Site 73
UniProt: Iron-sulfur 1 (4Fe-4S).
Metal-Binding-Site 79
UniProt: Iron-sulfur 1 (4Fe-4S).
Metal-Binding-Site 94
UniProt: Iron-sulfur 2 (4Fe-4S-S-AdoMet).
Mutagenesis-Variant 94 -> 101
[Cicchillo04a, UniProt11]
UniProt: Loss of 1 4Fe-4S cluster binding. Loss of activity.
Metal-Binding-Site 98
UniProt: Iron-sulfur 2 (4Fe-4S-S-AdoMet).
Metal-Binding-Site 101
UniProt: Iron-sulfur 2 (4Fe-4S-S-AdoMet).

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


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


Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

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

Cicchillo04a: 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

Cicchillo05: 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

Feng14: Feng Y, Cronan JE (2014). "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

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

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). "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

Gonidakis10a: 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

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

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

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

Patrick07: Patrick WM, Quandt EM, Swartzlander DB, Matsumura I (2007). "Multicopy suppression underpins metabolic evolvability." Mol Biol Evol 24(12);2716-22. PMID: 17884825

Reed93: 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.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

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

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms 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

Other References Related to Gene Regulation

MendozaVargas09: Mendoza-Vargas A, Olvera L, Olvera M, Grande R, Vega-Alvarado L, Taboada B, Jimenez-Jacinto V, Salgado H, Juarez K, Contreras-Moreira B, Huerta AM, Collado-Vides J, Morett E (2009). "Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli." PLoS One 4(10);e7526. PMID: 19838305

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 19.0 on Tue Oct 13, 2015, BIOCYC11A.