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Escherichia coli K-12 substr. MG1655 Enzyme: undecaprenyl diphosphate synthase



Gene: ispU Accession Numbers: G6092 (EcoCyc), b0174, ECK0173

Synonyms: uppS, rth, yaeS

Regulation Summary Diagram: ?

Subunit composition of undecaprenyl diphosphate synthase = [IspU]2
         subunit of undecaprenyl diphosphate synthase = IspU

Summary:
Undecaprenyl diphosphate (UPP) synthase catalyzes the condensation reactions resulting in the formation of UPP, a di-trans,poly-cis-undecaprenyl pyrophosphate that functions as the lipid carrier for bacterial cell wall carbohydrates. To generate UPP, eight isopentenyl diphosphate molecules are sequentially added to farnesyl diphosphate with cis stereochemistry [Baba85, Fujisaki86, Apfel99, Kato99].

Under various reaction conditions, the enzyme can produce C50 intermediates as well as larger C60-C75 products in addition to UPP. Triton X-100 activates the reaction by accelerating a step after the rate-limiting IPP condensation reactions [Pan00]. Site-directed mutagenesis of conserved residues identified amino acids that are required for catalysis and/or play a role in binding of IPP [Pan00a, Chen04b], and play a role in product chain length determination [Ko01]. Molecular dynamics simulations indicate that IspU is a highly flexible protein [Sinko11]. A conformational change of the enzyme during catalysis is thought to occur [Chen02c]. The length of a flexible loop region appears to regulate the conformational change [Chang03]. Single-turnover experiments support a concerted condensation-elimination mechanism for the enzyme [Lu10]. Substrate specificity has been studied, and the shape of both the active site and the product are predicted to determine the chain length of the product [Chen04b].

Crystal structures of IspU have been solved, revealing a possible reaction mechanism [Ko01, Chang03a, Chang04a, Guo05, Guo07, Sinko11]. UPP synthase appears to be a dimer in solution and in the crystal structure [Apfel99, Chang03a].

ispU is essential in E. coli [Kato99, Baba06]. Mutants with temperature-sensitive alleles of ispU are sensitive to low osmolarity of the growth medium and have a cell shape defect when grown at the restrictive temperature [Kato99]. Inhibitors of the enzyme have been investigated as novel antimicrobial agents [Peukert08]. ispU gene expression is repressed by MetJ, which appears to bind a site upstream of this gene [Marincs06].

rth: "RER two homologue" [Kato99]
isp: isoprenoid

Review: [Koyama99a]

Locations: cytosol

Map Position: [194,903 -> 195,664] (4.2 centisomes)
Length: 762 bp / 253 aa

Molecular Weight of Polypeptide: 28.444 kD (from nucleotide sequence), 29 kD (experimental) [Apfel99 ]

Unification Links: ASAP:ABE-0000595 , DIP:DIP-48251N , DisProt:DP00516 , EchoBASE:EB3113 , EcoGene:EG13329 , EcoliWiki:b0174 , ModBase:P60472 , OU-Microarray:b0174 , PortEco:ispU , PR:PRO_000024186 , Pride:P60472 , Protein Model Portal:P60472 , RefSeq:NP_414716 , RegulonDB:G6092 , SMR:P60472 , String:511145.b0174 , UniProt:P60472

Relationship Links: InterPro:IN-FAMILY:IPR001441 , InterPro:IN-FAMILY:IPR018520 , Panther:IN-FAMILY:PTHR10291 , PDB:Structure:1JP3 , PDB:Structure:1UEH , PDB:Structure:1V7U , PDB:Structure:1X06 , PDB:Structure:1X07 , PDB:Structure:1X08 , PDB:Structure:1X09 , PDB:Structure:2E98 , PDB:Structure:2E99 , PDB:Structure:2E9A , PDB:Structure:2E9C , PDB:Structure:2E9D , PDB:Structure:3QAS , PDB:Structure:3SGV , PDB:Structure:3SGX , PDB:Structure:3SH0 , PDB:Structure:3TH8 , PDB:Structure:4H2J , PDB:Structure:4H2M , PDB:Structure:4H2O , PDB:Structure:4H38 , PDB:Structure:4H3A , PDB:Structure:4H3C , Pfam:IN-FAMILY:PF01255 , Prosite:IN-FAMILY:PS01066

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0009252 - peptidoglycan biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA06, Kato99]
GO:0016094 - polyprenol biosynthetic process Inferred from experiment [Pan00]
GO:0043164 - Gram-negative-bacterium-type cell wall biogenesis Inferred from experiment [Kato99]
GO:0007049 - cell cycle Inferred by computational analysis [UniProtGOA11]
GO:0008360 - regulation of cell shape Inferred by computational analysis [UniProtGOA11]
GO:0051301 - cell division Inferred by computational analysis [UniProtGOA11]
GO:0071555 - cell wall organization Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0000287 - magnesium ion binding Inferred from experiment Inferred by computational analysis [GOA06, Chang03a]
GO:0008834 - di-trans,poly-cis-decaprenylcistransferase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, Pan00]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
GO:0016765 - transferase activity, transferring alkyl or aryl (other than methyl) groups Inferred by computational analysis [GOA01]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005737 - cytoplasm Inferred from experiment [Apfel99]
GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]

MultiFun Terms: metabolism biosynthesis of macromolecules (cellular constituents) murein (peptidoglycan)

Essentiality data for ispU knockouts: ?

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

Credits:
Last-Curated ? 18-Feb-2011 by Keseler I , SRI International


Enzymatic reaction of: di-trans,poly-cis-decaprenyl diphosphate:isopentenyl-diphosphate undecaprenylcistransferase (undecaprenyl diphosphate synthase)

Synonyms: di-trans,poly-cis-decaprenylcistransferase, di-trans,poly-cis-undecaprenyl-diphosphate synthase, bactoprenyl diphosphate synthase, undecaprenyl pyrophosphate synthase, UPP synthase

EC Number: 2.5.1.31

(2E,6E)-farnesyl diphosphate + 8 isopentenyl diphosphate <=> di-trans,octa-cis-undecaprenyl diphosphate + 8 diphosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

Reversibility of this reaction is unspecified.

In Pathways: polyisoprenoid biosynthesis (E. coli) , di-trans,poly-cis-undecaprenyl phosphate biosynthesis

Cofactors or Prosthetic Groups: Mg2+ [Guo05, Apfel99]

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
(2E,6E)-farnesyl diphosphate
0.4
[Teng11, BRENDA14]
(2E,6E)-farnesyl diphosphate
0.37, 0.44, 1.5, 1.57
1.23, 1.92, 1.98
[Guo05, BRENDA14]
(2E,6E)-farnesyl diphosphate
0.4
0.013
[Pan00, BRENDA14]
isopentenyl diphosphate
9.7, 11.5, 17.5, 291.0, 0.41
[Guo05, BRENDA14]
isopentenyl diphosphate
13.0
[Chen04b, BRENDA14]
isopentenyl diphosphate
16.2
[Pan00a, BRENDA14]
isopentenyl diphosphate
29.0
[Fujisaki86, BRENDA14]
isopentenyl diphosphate
4.1
[Pan00]
isopentenyl diphosphate
4.0
2.5
[Chang03a, BRENDA14]

pH(opt): 7.5 [BRENDA14, Fujisaki86], 7.5-9 [Fujisaki86]


Sequence Features

Feature Class Location Citations Comment
Metal-Binding-Site 26
[UniProt12]
UniProt: Magnesium.
Protein-Segment 26 -> 30
[UniProt12]
UniProt: Substrate binding; Sequence Annotation Type: region of interest.
Mutagenesis-Variant 26
[Pan00a, UniProt11]
Alternate sequence: D → A; UniProt: Great decrease in activity.
Active-Site 26
[UniProt10a]
Mutagenesis-Variant 31
[Chen02c, UniProt11]
Alternate sequence: W → F; UniProt: Decrease in activity; reduced affinity for decaprenyl diphosphate substrate analog.
Amino-Acid-Sites-That-Bind 31
[UniProt10]
UniProt: Decaprenyl diphosphate; Non-Experimental Qualifier: probable;
Amino-Acid-Sites-That-Bind 39
[UniProt12]
UniProt: Substrate.
Mutagenesis-Variant 43
[Chang03a, UniProt12]
Alternate sequence: H → A; UniProt: Great decreases the catalytic efficiency and the affinty for FPP and IPP.
Amino-Acid-Sites-That-Bind 43
[UniProt12]
UniProt: Substrate.
Mutagenesis-Variant 62
[Ko01, UniProt11]
Alternate sequence: I → A; UniProt: Formation predominantly of C(60) and C(65) polymers rather than the C(55) polymer.
Mutagenesis-Variant 69
[Ko01, UniProt11]
Alternate sequence: A → L; UniProt: Produces shorter polymers.
Amino-Acid-Site 69
[UniProt10a]
UniProt: Required for continued chain elongation; Sequence Annotation Type: site;
Protein-Segment 71 -> 73
[UniProt12]
UniProt: Substrate binding; Sequence Annotation Type: region of interest; Non-Experimental Qualifier: by similarity.
Mutagenesis-Variant 71
[Ko01, UniProt11]
Alternate sequence: S → A; UniProt: Decrease in activity.
Mutagenesis-Variant 73
[Ko01, Pan00a, UniProt11]
Alternate sequence: E → A; UniProt: Slight decrease in activity.
Active-Site 74
[UniProt12]
UniProt: Proton acceptor; Non-Experimental Qualifier: by similarity.
Mutagenesis-Variant 74
[Ko01, UniProt11]
Alternate sequence: N → A; UniProt: Decrease in activity.
Mutagenesis-Variant 75
[Ko01, Chen02c, UniProt11]
Alternate sequence: W → F; UniProt: Decrease in activity; reduced affinity for decaprenyl diphosphate substrate analog.
Alternate sequence: W → A; UniProt: Decrease in activity; reduced affinity for decaprenyl diphosphate substrate analog.
Amino-Acid-Sites-That-Bind 75
[UniProt10]
UniProt: Decaprenyl diphosphate; Non-Experimental Qualifier: probable;
Amino-Acid-Sites-That-Bind 77
[UniProt12]
UniProt: Substrate.
Mutagenesis-Variant 77
[Ko01, UniProt11]
Alternate sequence: R → A; UniProt: Decrease in activity.
Mutagenesis-Variant 81
[Ko01, UniProt11]
Alternate sequence: E → A; UniProt: Slight decrease in activity.
Mutagenesis-Variant 91
[Chen02c, UniProt11]
Alternate sequence: W → F; UniProt: Decrease in affinity for IPP.
Mutagenesis-Variant 103
[Ko01, UniProt11]
Alternate sequence: H → A; UniProt: No effect.
Mutagenesis-Variant 105
[Ko01, UniProt11]
Alternate sequence: V → A; UniProt: Formation predominantly of C(60), C(65) and C(70) polymers rather than the C(55) polymer.
Mutagenesis-Variant 137
[Ko01, UniProt11]
Alternate sequence: L → A; UniProt: Formation predominantly of a C(70) polymer rather than the C(55) polymer.
Amino-Acid-Site 137
[UniProt10a]
UniProt: Important for determining product length; Sequence Annotation Type: site;
Mutagenesis-Variant 143
[Ko01, UniProt11]
Alternate sequence: A → V; UniProt: No effect on polymer length.
Mutagenesis-Variant 149
[Chen02c, UniProt11]
Alternate sequence: W → F; UniProt: Decrease in affinity for IPP.
Mutagenesis-Variant 150
[Pan00a, UniProt11]
Alternate sequence: D → A; UniProt: Great decrease in affinity for the substrate.
Mutagenesis-Variant 190
[Pan00a, UniProt11]
Alternate sequence: D → A; UniProt: No effect.
Amino-Acid-Sites-That-Bind 194
[UniProt12]
UniProt: Substrate.
Mutagenesis-Variant 198
[Pan00a, UniProt11]
Alternate sequence: E → A; UniProt: No effect.
Mutagenesis-Variant 199
[Chang03a, UniProt12]
Alternate sequence: H → A; UniProt: Great decreases in the catalytic efficiency and in the affinity for IPP; when associated with A-213.
Metal-Binding-Site 199
[UniProt12]
UniProt: Magnesium.
Protein-Segment 200 -> 202
[UniProt12]
UniProt: Substrate binding; Sequence Annotation Type: region of interest.
Mutagenesis-Variant 207
[Chen02c, UniProt11]
Alternate sequence: W → F; UniProt: Decrease in affinity for both IPP and decaprenyl diphosphate substrate analog.
Mutagenesis-Variant 213
[Chang03a, Pan00a, UniProt12]
Alternate sequence: E → A; UniProt: Great decrease in activity; reduced affinity for IPP. Great decreases in the catalytic efficiency and in the affinity for IPP; when associated with A-199.
Metal-Binding-Site 213
[UniProt12]
UniProt: Magnesium.
Amino-Acid-Sites-That-Bind 213
[UniProt10a]
UniProt: Isopentenyl diphosphate;
Mutagenesis-Variant 218
[Pan00a, UniProt11]
Alternate sequence: D → A; UniProt: Slight decrease in activity.
Mutagenesis-Variant 221
[Chen02c, UniProt11]
Alternate sequence: W → F; UniProt: Decrease in affinity for IPP.
Mutagenesis-Variant 223
[Pan00a, UniProt11]
Alternate sequence: D → A; UniProt: No effect.


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
Markus Krummenacker on Tue Oct 14, 1997:
Gene object created from Blattner lab Genbank (v. M52) entry.


References

Apfel99: Apfel CM, Takacs B, Fountoulakis M, Stieger M, Keck W (1999). "Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene." J Bacteriol 1999;181(2);483-92. PMID: 9882662

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

Baba85: Baba T, Muth J, Allen CM (1985). "Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue." J Biol Chem 1985;260(19);10467-73. PMID: 3897217

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

Chang03: Chang SY, Chen YK, Wang AH, Liang PH (2003). "Identification of the active conformation and the importance of length of the flexible loop 72-83 in regulating the conformational change of undecaprenyl pyrophosphate synthase." Biochemistry 42(49);14452-9. PMID: 14661956

Chang03a: Chang SY, Ko TP, Liang PH, Wang AH (2003). "Catalytic mechanism revealed by the crystal structure of undecaprenyl pyrophosphate synthase in complex with sulfate, magnesium, and triton." J Biol Chem 278(31);29298-307. PMID: 12756244

Chang04a: Chang SY, Ko TP, Chen AP, Wang AH, Liang PH (2004). "Substrate binding mode and reaction mechanism of undecaprenyl pyrophosphate synthase deduced from crystallographic studies." Protein Sci 13(4);971-8. PMID: 15044730

Chen02c: Chen YH, Chen AP, Chen CT, Wang AH, Liang PH (2002). "Probing the conformational change of Escherichia coli undecaprenyl pyrophosphate synthase during catalysis using an inhibitor and tryptophan mutants." J Biol Chem 277(9);7369-76. PMID: 11744728

Chen02d: Chen AP, Chen YH, Liu HP, Li YC, Chen CT, Liang PH (2002). "Synthesis and application of a fluorescent substrate analogue to study ligand interactions for undecaprenyl pyrophosphate synthase." J Am Chem Soc 124(51);15217-24. PMID: 12487597

Chen04b: Chen AP, Chang SY, Lin YC, Sun YS, Chen CT, Wang AH, Liang PH (2004). "Substrate and product specificities of cis-type undecaprenyl pyrophosphate synthase." Biochem J Pt. PMID: 15447632

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

Fujisaki86: Fujisaki S, Nishino T, Katsuki H (1986). "Isoprenoid synthesis in Escherichia coli. Separation and partial purification of four enzymes involved in the synthesis." J Biochem (Tokyo) 1986;99(5);1327-37. PMID: 3519603

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."

Guo05: Guo RT, Ko TP, Chen AP, Kuo CJ, Wang AH, Liang PH (2005). "Crystal structures of undecaprenyl pyrophosphate synthase in complex with magnesium, isopentenyl pyrophosphate, and farnesyl thiopyrophosphate: roles of the metal ion and conserved residues in catalysis." J Biol Chem 280(21);20762-74. PMID: 15788389

Guo07: Guo RT, Cao R, Liang PH, Ko TP, Chang TH, Hudock MP, Jeng WY, Chen CK, Zhang Y, Song Y, Kuo CJ, Yin F, Oldfield E, Wang AH (2007). "Bisphosphonates target multiple sites in both cis- and trans-prenyltransferases." Proc Natl Acad Sci U S A 104(24);10022-7. PMID: 17535895

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

Kato99: Kato J, Fujisaki S, Nakajima K, Nishimura Y, Sato M, Nakano A (1999). "The Escherichia coli homologue of yeast RER2, a key enzyme of dolichol synthesis, is essential for carrier lipid formation in bacterial cell wall synthesis." J Bacteriol 1999;181(9);2733-8. PMID: 10217761

Ko01: Ko TP, Chen YK, Robinson H, Tsai PC, Gao YG, Chen AP, Wang AH, Liang PH (2001). "Mechanism of product chain length determination and the role of a flexible loop in Escherichia coli undecaprenyl-pyrophosphate synthase catalysis." J Biol Chem 276(50);47474-82. PMID: 11581264

Koyama99a: Koyama T (1999). "Molecular analysis of prenyl chain elongating enzymes." Biosci Biotechnol Biochem 63(10);1671-6. PMID: 10586494

Lu10: Lu YP, Liu HG, Teng KH, Liang PH (2010). "Mechanism of cis-prenyltransferase reaction probed by substrate analogues." Biochem Biophys Res Commun 400(4);758-62. PMID: 20828539

Marincs06: Marincs F, Manfield IW, Stead JA, McDowall KJ, Stockley PG (2006). "Transcript analysis reveals an extended regulon and the importance of protein-protein co-operativity for the Escherichia coli methionine repressor." Biochem J 396(2);227-34. PMID: 16515535

Pan00: Pan JJ, Chiou ST, Liang PH (2000). "Product distribution and pre-steady-state kinetic analysis of Escherichia coli undecaprenyl pyrophosphate synthase reaction." Biochemistry 39(35);10936-42. PMID: 10978182

Pan00a: Pan JJ, Yang LW, Liang PH (2000). "Effect of site-directed mutagenesis of the conserved aspartate and glutamate on E. coli undecaprenyl pyrophosphate synthase catalysis." Biochemistry 39(45);13856-61. PMID: 11076526

Peukert08: Peukert S, Sun Y, Zhang R, Hurley B, Sabio M, Shen X, Gray C, Dzink-Fox J, Tao J, Cebula R, Wattanasin S (2008). "Design and structure-activity relationships of potent and selective inhibitors of undecaprenyl pyrophosphate synthase (UPPS): tetramic, tetronic acids and dihydropyridin-2-ones." Bioorg Med Chem Lett 18(6);1840-4. PMID: 18295483

Sinko11: Sinko W, de Oliveira C, Williams S, Van Wynsberghe A, Durrant JD, Cao R, Oldfield E, McCammon JA (2011). "Applying molecular dynamics simulations to identify rarely sampled ligand-bound conformational states of undecaprenyl pyrophosphate synthase, an antibacterial target." Chem Biol Drug Des 77(6);412-20. PMID: 21294851

Teng11: Teng KH, Chen AP, Kuo CJ, Li YC, Liu HG, Chen CT, Liang PH (2011). "Fluorescent substrate analog for monitoring chain elongation by undecaprenyl pyrophosphate synthase in real time." Anal Biochem 417(1);136-41. PMID: 21704016

UniProt10: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

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.

UniProt12: UniProt Consortium (2012). "UniProt version 2012-11 released on 2012-11-26 00:00:00." Database.

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

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 18.5 on Wed Nov 26, 2014, biocyc13.