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) is involved in the sixth step of the methylerythritol phosphate (MEP) pathway. IspG catalyzes 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 as-yet unidentified 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, Zepeck05]. Higher IspG activity was observed under new assay conditions which detected the [4Fe-4S] successfully [Xiao09]. Mutations in cysteine residues responsible for Fe-S cluster coordination abolish IspG's enzymatic activity [Zepeck05]. Similarly, flux through the entire methylerythritol phosphate pathway depends on the presence of flavodoxin I, which may supply reducing equivalents necessary to reduce the Fe-S cluster and yield an active enzyme [Puan05]. Overexpression of IspG was found to significantly increase downstream isoprenoid production [Zhou12].

An ispG mutant shows a defect in activation of human Vgamma9/Vdelta2 T lymphocytes due to a defect in production of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), an antigenic compound that accumulates in an ispH mutant [Altincicek01a, Hintz01].

ispG is essential [Baker92, Altincicek01, SauretGueto03].

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

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)

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:0006744 - ubiquinone biosynthetic process Inferred from experiment [Zepeck05]
GO:0008299 - isoprenoid biosynthetic process Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0016114 - terpenoid biosynthetic process Inferred by computational analysis [GOA06, GOA01a]
GO:0019288 - isopentenyl diphosphate biosynthetic process, methylerythritol 4-phosphate pathway Inferred by computational analysis [UniProtGOA12]
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, GOA01, 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 08-Nov-2005 by Keseler I , SRI International
Curated 04-May-2007 by Shearer A , SRI International
Last-Curated ? 20-Aug-2013 by Kubo A , 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

Cofactors or Prosthetic Groups: Mn2+ [Zepeck05]

Kinetic Parameters:

Km (μM)

pH(opt): 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, UniProt11a]
UniProt: Loss of activity.
Mutagenesis-Variant 328
[SauretGueto03, UniProt11a]
UniProt: Loss of activity.
Mutagenesis-Variant 335
[SauretGueto03, UniProt11a]
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

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

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."

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

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

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

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

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

UniProt11a: 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

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

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

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 19.0 on Sat May 23, 2015, biocyc11.