Escherichia coli K-12 substr. MG1655 Enzyme: 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase

Gene: ispG Accession Numbers: EG10370 (EcoCyc), b2515, ECK2511

Synonyms: gcpE

Regulation Summary Diagram: ?

Regulation summary diagram for ispG

1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG) catalyzes the sixth step of the mevalonate-independent methylerythritol phosphate (MEP) pathway, the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate [Campos01, Altincicek01, Hecht01].

IspG activity depends on additional proteins, most likely involved in the oxidation portion of the reaction [Rohdich03]. IspG contains a [4Fe-4S] cluster that is required for its activity [Seemann02, Seemann05, Zepeck05]. Mutations in cysteine residues responsible for Fe-S cluster coordination abolish IspG's enzymatic activity [Zepeck05]. In vitro, ErpA can transfer a [4Fe-4S] cluster to apo-IspG [Loiseau07]. Increased IspG activity was observed under new assay conditions [Xiao09]. Flux through the entire MEP pathway was shown to depend on the presence of flavodoxin 1, which may supply reducing equivalents necessary to reduce the Fe-S cluster and yield an active enzyme [Puan05].

A new reaction mechanism was recently proposed [Wang10]. Further studies on the reaction mechanism and reaction intermediate have been published [Xiao10, Xiao11, Wang11a], and potential reaction mechanisms are reviewed in [Wang14a].

An ispG mutant does not activate human γδ T lymphocytes due to its defect in production of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). This intermediate in the MEP pathway accumulates in an ispH mutant [Altincicek01a, Hintz01].

ispG is essential [Baker92, Altincicek01, SauretGueto03]. Missense mutants which are defective in the MEP pathway [SauretGueto03], as well as a conditional amber mutant [Herring04] have been isolated.

Since the enzymes of the MEP pathway are not found in humans, they have attracted interest for their potential as anti-infective drug targets. A MEP pathway inhibitor has been identified and found to bind IspG [Nakagawa13].

Review: [Wang14a]

Citations: [McAteer01, Amslinger02, Nyland09, Zhou12, Liu12]

Gene Citations: [Vogel03]

Locations: cytosol

Map Position: [2,638,708 <- 2,639,826] (56.87 centisomes, 205°)
Length: 1119 bp / 372 aa

Molecular Weight of Polypeptide: 40.684 kD (from nucleotide sequence), 40.0 kD (experimental) [Rohdich03 ]

Unification Links: ASAP:ABE-0008281 , CGSC:32909 , DIP:DIP-35832N , EchoBASE:EB0365 , EcoGene:EG10370 , EcoliWiki:b2515 , ModBase:P62620 , OU-Microarray:b2515 , PortEco:ispG , PR:PRO_000023039 , Pride:P62620 , Protein Model Portal:P62620 , RefSeq:NP_417010 , RegulonDB:EG10370 , SMR:P62620 , String:511145.b2515 , UniProt:P62620

Relationship Links: InterPro:IN-FAMILY:IPR004588 , InterPro:IN-FAMILY:IPR011005 , InterPro:IN-FAMILY:IPR013785 , InterPro:IN-FAMILY:IPR016425 , Pfam:IN-FAMILY:PF04551

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0019288 - isopentenyl diphosphate biosynthetic process, methylerythritol 4-phosphate pathway Inferred from experiment Inferred by computational analysis [UniProtGOA12, Altincicek01, Campos01]
GO:0008299 - isoprenoid biosynthetic process Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0016114 - terpenoid biosynthetic process Inferred by computational analysis [GOA06, GOA01a]
GO:0044237 - cellular metabolic process Inferred by computational analysis [GOA01a]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11a, GOA01a]
Molecular Function: GO:0005515 - protein binding Inferred from experiment [Rajagopala14, Arifuzzaman06]
GO:0046429 - 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, Zepeck05]
GO:0046872 - metal ion binding Inferred from experiment Inferred by computational analysis [UniProtGOA11a, Zepeck05]
GO:0051539 - 4 iron, 4 sulfur cluster binding Inferred from experiment Inferred by computational analysis [UniProtGOA11a, GOA06, Seemann02]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01a]
GO:0005506 - iron ion binding Inferred by computational analysis [GOA01a]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11a]
GO:0051536 - iron-sulfur cluster binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05]

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

Essentiality data for ispG knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox No 37 Aerobic 7   No [Baba06, Comment 1]

Curated 04-May-2007 by Shearer A , SRI International
Curated 20-Aug-2013 by Kubo A , SRI International
Last-Curated ? 28-May-2015 by Keseler I , SRI International

Enzymatic reaction of: 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase

EC Number:

1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate + an oxidized flavodoxin + H2O + H+ <=> 2-C-methyl-D-erythritol-2,4-cyclodiphosphate + a reduced flavodoxin

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 physiologically favored in the opposite direction.

In Pathways: methylerythritol phosphate pathway I

Citations: [Nyland09]

Cofactors or Prosthetic Groups: a [4Fe-4S] iron-sulfur cluster [Seemann02], Mn2+ [Zepeck05]

Kinetic Parameters:

Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)

pH(opt) (reverse direction): 9 [Zepeck05]

Sequence Features

Protein sequence of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase with features indicated

Feature Class Location Citations Comment
Metal-Binding-Site 270
UniProt: Iron-sulfur (4Fe-4S); Non-Experimental Qualifier: by similarity.
Metal-Binding-Site 273
UniProt: Iron-sulfur (4Fe-4S); Non-Experimental Qualifier: by similarity.
Metal-Binding-Site 305
UniProt: Iron-sulfur (4Fe-4S); Non-Experimental Qualifier: by similarity.
Metal-Binding-Site 312
UniProt: Iron-sulfur (4Fe-4S); Non-Experimental Qualifier: by similarity.
Mutagenesis-Variant 322
[SauretGueto03, UniProt11]
UniProt: Loss of activity.
Mutagenesis-Variant 328
[SauretGueto03, UniProt11]
UniProt: Loss of activity.
Mutagenesis-Variant 335
[SauretGueto03, UniProt11]
UniProt: Loss of activity.

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


10/20/97 Gene b2515 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10370; confirmed by SwissProt match.


Altincicek01: Altincicek B, Kollas AK, Sanderbrand S, Wiesner J, Hintz M, Beck E, Jomaa H (2001). "GcpE is involved in the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli." J Bacteriol 183(8);2411-6. PMID: 11274098

Altincicek01a: Altincicek B, Moll J, Campos N, Foerster G, Beck E, Hoeffler JF, Grosdemange-Billiard C, Rodriguez-Concepcion M, Rohmer M, Boronat A, Eberl M, Jomaa H (2001). "Cutting edge: human gamma delta T cells are activated by intermediates of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis." J Immunol 166(6);3655-8. PMID: 11238603

Amslinger02: Amslinger S, Kis K, Hecht S, Adam P, Rohdich F, Arigoni D, Bacher A, Eisenreich W (2002). "Biosynthesis of terpenes. Preparation of (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate, an intermediate of the deoxyxylulose phosphate pathway." J Org Chem 67(13);4590-4. PMID: 12076162

Arifuzzaman06: Arifuzzaman M, Maeda M, Itoh A, Nishikata K, Takita C, Saito R, Ara T, Nakahigashi K, Huang HC, Hirai A, Tsuzuki K, Nakamura S, Altaf-Ul-Amin M, Oshima T, Baba T, Yamamoto N, Kawamura T, Ioka-Nakamichi T, Kitagawa M, Tomita M, Kanaya S, Wada C, Mori H (2006). "Large-scale identification of protein-protein interaction of Escherichia coli K-12." Genome Res 16(5);686-91. PMID: 16606699

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

Baker92: Baker J, Franklin DB, Parker J (1992). "Sequence and characterization of the gcpE gene of Escherichia coli." FEMS Microbiol Lett 73(1-2);175-80. PMID: 1521767

Campos01: Campos N, Rodriguez-Concepcion M, Seemann M, Rohmer M, Boronat A (2001). "Identification of gcpE as a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis in Escherichia coli." FEBS Lett 488(3);170-3. PMID: 11163766

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

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."

Hecht01: Hecht S, Eisenreich W, Adam P, Amslinger S, Kis K, Bacher A, Arigoni D, Rohdich F (2001). "Studies on the nonmevalonate pathway to terpenes: the role of the GcpE (IspG) protein." Proc Natl Acad Sci U S A 98(26);14837-42. PMID: 11752431

Herring04: Herring CD, Blattner FR (2004). "Conditional lethal amber mutations in essential Escherichia coli genes." J Bacteriol 186(9);2673-81. PMID: 15090508

Hintz01: Hintz M, Reichenberg A, Altincicek B, Bahr U, Gschwind RM, Kollas AK, Beck E, Wiesner J, Eberl M, Jomaa H (2001). "Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli." FEBS Lett 509(2);317-22. PMID: 11741609

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

Liu12: Liu YL, Guerra F, Wang K, Wang W, Li J, Huang C, Zhu W, Houlihan K, Li Z, Zhang Y, Nair SK, Oldfield E (2012). "Structure, function and inhibition of the two- and three-domain 4Fe-4S IspG proteins." Proc Natl Acad Sci U S A 109(22);8558-63. PMID: 22586085

Loiseau07: Loiseau L, Gerez C, Bekker M, Ollagnier-de Choudens S, Py B, Sanakis Y, Teixeira de Mattos J, Fontecave M, Barras F (2007). "ErpA, an iron sulfur (Fe S) protein of the A-type essential for respiratory metabolism in Escherichia coli." Proc Natl Acad Sci U S A 104(34);13626-31. PMID: 17698959

LopezCampistrou05: Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005). "Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth." Mol Cell Proteomics 4(8);1205-9. PMID: 15911532

McAteer01: McAteer S, Coulson A, McLennan N, Masters M (2001). "The lytB gene of Escherichia coli is essential and specifies a product needed for isoprenoid biosynthesis." J Bacteriol 183(24);7403-7. PMID: 11717301

Nakagawa13: Nakagawa K, Takada K, Imamura N (2013). "Probable Novel MEP Pathway Inhibitor and Its Binding Protein, IspG." Biosci Biotechnol Biochem 77(7);1449-54. PMID: 23832336

Nyland09: Nyland RL, Xiao Y, Liu P, Freel Meyers CL (2009). "IspG converts an epoxide substrate analogue to (E)-4-hydroxy-3-methylbut-2-enyl diphosphate: implications for IspG catalysis in isoprenoid biosynthesis." J Am Chem Soc 131(49);17734-5. PMID: 19919056

Puan05: Puan KJ, Wang H, Dairi T, Kuzuyama T, Morita CT (2005). "fldA is an essential gene required in the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis." FEBS Lett 579(17);3802-6. PMID: 15978585

Rajagopala14: Rajagopala SV, Sikorski P, Kumar A, Mosca R, Vlasblom J, Arnold R, Franca-Koh J, Pakala SB, Phanse S, Ceol A, Hauser R, Siszler G, Wuchty S, Emili A, Babu M, Aloy P, Pieper R, Uetz P (2014). "The binary protein-protein interaction landscape of Escherichia coli." Nat Biotechnol 32(3);285-90. PMID: 24561554

Rohdich03: Rohdich F, Zepeck F, Adam P, Hecht S, Kaiser J, Laupitz R, Grawert T, Amslinger S, Eisenreich W, Bacher A, Arigoni D (2003). "The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein." Proc Natl Acad Sci U S A 100(4);1586-91. PMID: 12571359

SauretGueto03: Sauret-Gueto S, Ramos-Valdivia A, Ibanez E, Boronat A, Rodriguez-Concepcion M (2003). "Identification of lethal mutations in Escherichia coli genes encoding enzymes of the methylerythritol phosphate pathway." Biochem Biophys Res Commun 307(2);408-15. PMID: 12859972

Seemann02: Seemann M, Bui BT, Wolff M, Tritsch D, Campos N, Boronat A, Marquet A, Rohmer M (2002). "Isoprenoid biosynthesis through the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) is a [4Fe-4S] protein." Angew Chem Int Ed Engl 41(22);4337-9. PMID: 12434382

Seemann05: Seemann M, Wegner P, Schunemann V, Bui BT, Wolff M, Marquet A, Trautwein AX, Rohmer M (2005). "Isoprenoid biosynthesis in chloroplasts via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) from Arabidopsis thaliana is a [4Fe-4S] protein." J Biol Inorg Chem 10(2);131-7. PMID: 15650872

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

UniProt12: UniProt Consortium (2012). "UniProt version 2012-09 released on 2012-09-12 00:00:00." Database.

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

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Vogel03: Vogel J, Bartels V, Tang TH, Churakov G, Slagter-Jager JG, Huttenhofer A, Wagner EG (2003). "RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria." Nucleic Acids Res 31(22);6435-43. PMID: 14602901

Wang10: Wang W, Li J, Wang K, Huang C, Zhang Y, Oldfield E (2010). "Organometallic mechanism of action and inhibition of the 4Fe-4S isoprenoid biosynthesis protein GcpE (IspG)." Proc Natl Acad Sci U S A 107(25);11189-93. PMID: 20534554

Wang11a: Wang W, Wang K, Li J, Nellutla S, Smirnova TI, Oldfield E (2011). "An ENDOR and HYSCORE investigation of a reaction intermediate in IspG (GcpE) catalysis." J Am Chem Soc 133(22);8400-3. PMID: 21574560

Wang14a: Wang W, Oldfield E (2014). "Bioorganometallic chemistry with IspG and IspH: structure, function, and inhibition of the [Fe(4)S(4)] proteins involved in isoprenoid biosynthesis." Angew Chem Int Ed Engl 53(17);4294-310. PMID: 24481599

Xiao09: Xiao Y, Zahariou G, Sanakis Y, Liu P (2009). "IspG enzyme activity in the deoxyxylulose phosphate pathway: roles of the iron-sulfur cluster." Biochemistry 48(44);10483-5. PMID: 19821611

Xiao10: Xiao Y, Nyland RL, Meyers CL, Liu P (2010). "Methylerythritol cyclodiphosphate (MEcPP) in deoxyxylulose phosphate pathway: synthesis from an epoxide and mechanisms." Chem Commun (Camb) 46(38);7220-2. PMID: 20820543

Xiao11: Xiao Y, Rooker D, You Q, Meyers CL, Liu P (2011). "IspG-catalyzed positional isotopic exchange in methylerythritol cyclodiphosphate of the deoxyxylulose phosphate pathway: mechanistic implications." Chembiochem 12(4);527-30. PMID: 22238143

Zepeck05: Zepeck F, Grawert T, Kaiser J, Schramek N, Eisenreich W, Bacher A, Rohdich F (2005). "Biosynthesis of Isoprenoids. Purification and Properties of IspG Protein from Escherichia coli." J Org Chem 70(23);9168-74. PMID: 16268586

Zhou12: Zhou K, Zou R, Stephanopoulos G, Too HP (2012). "Metabolite profiling identified methylerythritol cyclodiphosphate efflux as a limiting step in microbial isoprenoid production." PLoS One 7(11);e47513. PMID: 23133596

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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