Escherichia coli K-12 substr. MG1655 Enzyme: 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase

Gene: ispF Accession Numbers: EG11816 (EcoCyc), b2746, ECK2741

Synonyms: ygbB

Regulation Summary Diagram: ?

Regulation summary diagram for ispF

Subunit composition of 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase = [IspF]3
         2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase monomer = IspF

2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) is involved in the fifth step of the methylerythritol phosphate pathway. IspF catalyzes the Mn2+- or Mg2+-dependent conversion of 2-phospho-4-(cytidine 5'diphospho)-2-C-methyl-D-erythritol into 2-C-methyl-D-erythritol-2,4-cyclodiphosphate [Herz00, Takagi00].

Substantial structural research has been done on IspF. Crystal structures of IspF on its own and bound to subtrates reveal that it is a trimer, with a central hydrophobic cavity and three active sites located between the subunits. One Zn2+ is bound at each active site [Steinbacher02, Richard02, Kemp02].

IspF binds various isoprenoid compounds, downstream products derived from the methylerythritol phosphate pathway, in its hydrophobic central cavity. The isoprenoids isopentenyl diphosphate/dimethylallyl diphosphate, geranyl diphosphate, and (2E,6E)-farnesyl diphosphate appear bound to IspF in a 1:4:2 ratio [Kemp05]. Although Arg-142 in each IspF monomer forms a hydrophobic bond with the diphosphate group in the bound isoprenoid, these arginines are not required for isoprenoid binding [Sgraja05]. IspF is not regulated by the downstream metabolites by negative feedback inhibition. 2C-methyl-D-erythritol 4-phosphate (MEP), the first committed MEP pathway intermediate, activates and sustains IspF activity. IspF-MEP complex is inhibited by farnesyl diphosphate [Bitok12].

ispF is an essential gene [Campos01c, Freiberg01, Campbell02b]. Loss of IspF function leads to growth defects, filamentous growth, and a sensitivity to antibiotics that target the cell wall [Campbell02b].

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. Both computational and high-throughput experimental methods have been used to attempt to identify inhibitors of IspF [Ramsden09, Illarionova06].

Locations: cytosol

Map Position: [2,869,323 <- 2,869,802] (61.84 centisomes, 223°)
Length: 480 bp / 159 aa

Molecular Weight of Polypeptide: 16.898 kD (from nucleotide sequence), 17 kD (experimental) [Herz00 ]

Unification Links: ASAP:ABE-0009013 , DIP:DIP-48029N , EchoBASE:EB1763 , EcoGene:EG11816 , EcoliWiki:b2746 , ModBase:P62617 , OU-Microarray:b2746 , PortEco:ispF , PR:PRO_000023038 , Protein Model Portal:P62617 , RefSeq:NP_417226 , RegulonDB:EG11816 , SMR:P62617 , String:511145.b2746 , UniProt:P62617

Relationship Links: InterPro:IN-FAMILY:IPR003526 , InterPro:IN-FAMILY:IPR020555 , PDB:Structure:1GX1 , PDB:Structure:1H47 , PDB:Structure:1H48 , PDB:Structure:1JY8 , PDB:Structure:1KNJ , PDB:Structure:1KNK , PDB:Structure:1U3L , PDB:Structure:1U3P , PDB:Structure:1U40 , PDB:Structure:1U43 , PDB:Structure:1YQN , PDB:Structure:2AMT , PDB:Structure:2GZL , PDB:Structure:3ELC , PDB:Structure:3EOR , PDB:Structure:3ERN , PDB:Structure:3ESJ , PDB:Structure:3FBA , Pfam:IN-FAMILY:PF02542 , Prosite:IN-FAMILY:PS01350

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0006744 - ubiquinone biosynthetic process Inferred from experiment [Herz00]
GO:0008299 - isoprenoid biosynthetic process Inferred by computational analysis [UniProtGOA11a]
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]
Molecular Function: GO:0008270 - zinc ion binding Inferred from experiment [Steinbacher02]
GO:0008685 - 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, Herz00]
GO:0030145 - manganese ion binding Inferred from experiment [Herz00]
GO:0042802 - identical protein binding Inferred from experiment [Steinbacher02]
GO:0046872 - metal ion binding Inferred from experiment Inferred by computational analysis [UniProtGOA11a, Herz00]
GO:0016829 - lyase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]

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

Essentiality data for ispF knockouts: ?

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

Curated 02-May-2007 by Shearer A , SRI International
Last-Curated ? 19-Aug-2013 by Kubo A , SRI International

Enzymatic reaction of: 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase

Synonyms: MECDP synthase, MEC synthase

EC Number:

2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol <=> CMP + 2-C-methyl-D-erythritol-2,4-cyclodiphosphate

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: methylerythritol phosphate pathway I

IspF can also convert 4-diphosphocytidyl-2-C-methyl-D-erythritol (the product of the IspD enzyme in the pathway) into 2-C-methyl-D-erythritol 3,4-cyclomonophosphate, but this compound does not appear to have any physiological relevance [Herz00].

Cofactors or Prosthetic Groups: Mn2+ [Herz00]

Kinetic Parameters:

Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
[Geist10, BRENDA14]

Sequence Features

Protein sequence of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase monomer with features indicated

Feature Class Location Attached Group Citations Comment
Metal-Binding-Site 8  
UniProt: Divalent metal cation.
Protein-Segment 8 -> 10  
UniProt: Substrate binding; Sequence Annotation Type: region of interest.
Mutagenesis-Variant 8  
[Steinbacher02, UniProt11]
UniProt: Loss of activity.
Metal-Binding-Site 8, 10, 42 Mn2+
Metal-Binding-Site 10  
UniProt: Divalent metal cation.
Amino-Acid-Site 34  
UniProt: Transition state stabilizer; Sequence Annotation Type: site.
Protein-Segment 34 -> 35  
UniProt: Substrate binding; Sequence Annotation Type: region of interest.
Protein-Segment 38 -> 46  
UniProt: Substrate binding; Sequence Annotation Type: region of interest.
Metal-Binding-Site 42  
UniProt: Divalent metal cation.
Mutagenesis-Variant 42  
[Steinbacher02, UniProt11]
UniProt: Loss of activity.
Protein-Segment 56 -> 58  
UniProt: Substrate binding; Sequence Annotation Type: region of interest.
Mutagenesis-Variant 56  
[Steinbacher02, UniProt11]
UniProt: 35% decrease of activity.
Protein-Segment 61 -> 65  
UniProt: Substrate binding; Sequence Annotation Type: region of interest.
Amino-Acid-Sites-That-Bind 65  
UniProt: Substrate; via carbonyl oxygen.
Protein-Segment 100 -> 106  
UniProt: Substrate binding; Sequence Annotation Type: region of interest.
Protein-Segment 131 -> 135  
UniProt: Substrate binding; Sequence Annotation Type: region of interest.
Amino-Acid-Site 133  
UniProt: Transition state stabilizer; Sequence Annotation Type: site.
Amino-Acid-Sites-That-Bind 139  
UniProt: Substrate; via carbonyl oxygen.
Amino-Acid-Sites-That-Bind 142  
UniProt: Substrate.
Mutagenesis-Variant 142  
[Sgraja05, UniProt12b]
UniProt: Little effect on the overall structure; when associated with L-144.
Mutagenesis-Variant 144  
[Sgraja05, UniProt12b]
UniProt: Little effect on the overall structure; when associated with M-142.

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


10/20/97 Gene b2746 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11816; 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

Bitok12: Bitok JK, Meyers CF (2012). "2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity." ACS Chem Biol 7(10);1702-10. PMID: 22839733

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014."

Campbell02b: Campbell TL, Brown ED (2002). "Characterization of the depletion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase in Escherichia coli and Bacillus subtilis." J Bacteriol 184(20);5609-18. PMID: 12270818

Campos01c: Campos N, Rodriguez-Concepcion M, Sauret-Gueto S, Gallego F, Lois LM, Boronat A (2001). "Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis." Biochem J 353(Pt 1);59-67. PMID: 11115399

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

Freiberg01: Freiberg C, Wieland B, Spaltmann F, Ehlert K, Brotz H, Labischinski H (2001). "Identification of novel essential Escherichia coli genes conserved among pathogenic bacteria." J Mol Microbiol Biotechnol 3(3);483-9. PMID: 11361082

Geist10: Geist JG, Lauw S, Illarionova V, Illarionov B, Fischer M, Grawert T, Rohdich F, Eisenreich W, Kaiser J, Groll M, Scheurer C, Wittlin S, Alonso-Gomez JL, Schweizer WB, Bacher A, Diederich F (2010). "Thiazolopyrimidine inhibitors of 2-methylerythritol 2,4-cyclodiphosphate synthase (IspF) from Mycobacterium tuberculosis and Plasmodium falciparum." ChemMedChem 5(7);1092-101. PMID: 20480490

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."

Herz00: Herz S, Wungsintaweekul J, Schuhr CA, Hecht S, Luttgen H, Sagner S, Fellermeier M, Eisenreich W, Zenk MH, Bacher A, Rohdich F (2000). "Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate to 2C-methyl-D-erythritol 2,4-cyclodiphosphate." Proc Natl Acad Sci U S A 2000;97(6);2486-90. PMID: 10694574

Illarionova06: Illarionova V, Kaiser J, Ostrozhenkova E, Bacher A, Fischer M, Eisenreich W, Rohdich F (2006). "Nonmevalonate terpene biosynthesis enzymes as antiinfective drug targets: substrate synthesis and high-throughput screening methods." J Org Chem 71(23);8824-34. PMID: 17081012

Kemp02: Kemp LE, Bond CS, Hunter WN (2002). "Structure of 2C-methyl-D-erythritol 2,4- cyclodiphosphate synthase: an essential enzyme for isoprenoid biosynthesis and target for antimicrobial drug development." Proc Natl Acad Sci U S A 99(10);6591-6. PMID: 11997478

Kemp05: Kemp LE, Alphey MS, Bond CS, Ferguson MA, Hecht S, Bacher A, Eisenreich W, Rohdich F, Hunter WN (2005). "The identification of isoprenoids that bind in the intersubunit cavity of Escherichia coli 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase by complementary biophysical methods." Acta Crystallogr D Biol Crystallogr 61(Pt 1);45-52. PMID: 15608374

Ramsden09: Ramsden NL, Buetow L, Dawson A, Kemp LA, Ulaganathan V, Brenk R, Klebe G, Hunter WN (2009). "A structure-based approach to ligand discovery for 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase: a target for antimicrobial therapy." J Med Chem 52(8);2531-42. PMID: 19320487

Richard02: Richard SB, Ferrer JL, Bowman ME, Lillo AM, Tetzlaff CN, Cane DE, Noel JP (2002). "Structure and mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase. An enzyme in the mevalonate-independent isoprenoid biosynthetic pathway." J Biol Chem 277(10);8667-72. PMID: 11786530

Sgraja05: Sgraja T, Kemp LE, Ramsden N, Hunter WN (2005). "A double mutation of Escherichia coli2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase disrupts six hydrogen bonds with, yet fails to prevent binding of, an isoprenoid diphosphate." Acta Crystallograph Sect F Struct Biol Cryst Commun 61(Pt 7);625-9. PMID: 16511114

Steinbacher02: Steinbacher S, Kaiser J, Wungsintaweekul J, Hecht S, Eisenreich W, Gerhardt S, Bacher A, Rohdich F (2002). "Structure of 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase involved in mevalonate-independent biosynthesis of isoprenoids." J Mol Biol 2002;316(1);79-88. PMID: 11829504

Takagi00: Takagi M, Kuzuyama T, Kaneda K, Watanabe H, Dairi T, Seto H (2000). "Studies on the nonmevalonate pathway: formation of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate from 2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol." Tetrahedron Lett. 41:3395-3398.

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

UniProt12b: UniProt Consortium (2012). "UniProt version 2012-11 released on 2012-11-26 00:00:00." Database.

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

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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 Mon Mar 30, 2015, biocyc14.