Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
twitter

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



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

Synonyms: lip

Regulation Summary Diagram: ?

Subunit composition of lipoate synthase = [LipA]2
         lipoate synthase monomer = LipA

Summary:
LipA is required for the step of lipoate biosynthesis at which sulfur is inserted into the octanoyl side-chain of an octanoylated E2 domain, to form the lipoate moiety [Reed93, Miller00a]. Lipoate is an important cofactor of several important enzyme complexes, such as pyruvate dehydrogenase [Herbert75, Stepp81, Reed93], alpha-ketoglutarate dehydrogenase [Herbert75, Stepp81, Reed93], and the glycine cleavage system [Vanden91, Reed93].

Lipoate synthase is a LipA homodimer with two (4Fe-4S) iron-sulfur clusters per protein dimer under anaerobic conditions, and these clusters are oxidized to the (2Fe-2S) state in air [OllagnierDe00, Ollagnierde99]. This arrangement of dimer interface-spanning, oxygen-sensitive iron-sulfur clusters is characteristic of S-adenosylmethionine-utilizing enzymes that exhibit a radical-mediated cleavage mechanism [Duin97]. The catalytic mechanisms of deoxyadenosine radical iron-sulfur enzymes are presented [Frey01]. The enzyme uses octanoyl side chains (but not free octanoate) as substrate and also uses S-adenosyl methionine [Miller00a]. Enzyme activity has been observed in vitro [Miller00a].

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]. Allele-specific effects on lipoic acid biosynthesis (at the sulfur insertion step) have been observed [Hayden93]. The lipoic acid auxotrophy of a lipA mutant is rescued by 6-thiooctanoic acid or 8-thiooctanoic acid [Reed93]. LipA and LipB are also necessary for anaerobic glycine cleavage system activity [Reed93]. The lipoic acid auxotrophy of a lipA mutant is enhanced by an slr-1 (selenolipoic acid-resistant) mutation [Reed94] and complemented by an slr-7 mutation [Reed94], which is a lip duplication [Jordan02].

The purification of overproduced LipA is described [Reed93, Ollagnierde99]. LipA has also been fused to a 6-His tag and purification of the fusion protein is described [Miller00a].

The site of translation initiation has been determined [Reed93].

Regulation has been described [Inoue02a].

Reviews: [Booker07, Frey01, Marquet01].

Citations: [Herbert68, Chang91a, Creaghan78]

Locations: cytosol

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

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

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 , 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: ?

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, Cicchillo04a]
GO:0055114 - oxidation-reduction process Inferred from experiment [Reed93]
Molecular Function: GO:0003826 - alpha-ketoacid dehydrogenase activity Inferred from experiment [Reed93]
GO:0016992 - lipoate synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Cicchillo04a]
GO:0051539 - 4 iron, 4 sulfur cluster binding Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA06, GOA01, 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, Cicchillo04a]
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]

Enzymatic reaction of: lipoate synthase

Synonyms: lipoic acid 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] <=> a [lipoyl-carrier protein] N6-lipoyl-L-lysine + 2 5'-deoxyadenosine + 2 L-methionine + 2 an unsulfurated [sulfur carrier]

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


Sequence Features

Feature Class Location Citations Comment
Sequence-Conflict 35
[Reed93, Hayden92, UniProt10]
Alternate sequence: L → V; UniProt: (in Ref. 1 and 2);
Mutagenesis-Variant 68 -> 79
[Cicchillo04b, UniProt11]
Alternate sequence: CEEASCPNLAEC → AEEASAPNLAEA; UniProt: Loss of 1 4Fe-4S cluster binding. Loss of activity.
Metal-Binding-Site 68
[UniProt10]
UniProt: Iron-sulfur 1 (4Fe-4S);
Metal-Binding-Site 73
[UniProt10]
UniProt: Iron-sulfur 1 (4Fe-4S);
Metal-Binding-Site 79
[UniProt10]
UniProt: Iron-sulfur 1 (4Fe-4S);
Mutagenesis-Variant 94 -> 101
[Cicchillo04b, UniProt11]
Alternate sequence: CTRRCPFC → ATRRAPFA; UniProt: Loss of 1 4Fe-4S cluster binding. Loss of activity.
Metal-Binding-Site 94
[UniProt10]
UniProt: Iron-sulfur 2 (4Fe-4S-S-AdoMet);
Metal-Binding-Site 98
[UniProt10]
UniProt: Iron-sulfur 2 (4Fe-4S-S-AdoMet);
Metal-Binding-Site 101
[UniProt10]
UniProt: Iron-sulfur 2 (4Fe-4S-S-AdoMet);


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

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


References

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

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

Chang91a: 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

Cicchillo04a: 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

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

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

Duin97: Duin EC, Lafferty ME, Crouse BR, Allen RM, Sanyal I, Flint DH, Johnson MK (1997). "[2Fe-2S] to [4Fe-4S] cluster conversion in Escherichia coli biotin synthase." Biochemistry 36(39);11811-20. PMID: 9305972

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."

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

Inoue02a: 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

Jordan02: Jordan SW, Cronan JE (2002). "Chromosomal amplification of the Escherichia coli lipB region confers high-level resistance to selenolipoic acid." J Bacteriol 184(19);5495-501. PMID: 12218038

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

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

Miller00a: 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

Reed94: Reed KE, Morris TW, Cronan JE (1994). "Mutants of Escherichia coli K-12 that are resistant to a selenium analog of lipoic acid identify unknown genes in lipoate metabolism." Proc Natl Acad Sci U S A 91(9);3720-4. PMID: 8170976

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

UniProt10: 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.

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

Other References Related to Gene Regulation

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

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

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


Report Errors or Provide Feedback
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 18.5 on Mon Nov 24, 2014, biocyc13.