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Escherichia coli K-12 substr. MG1655 Enzyme: dTDP-glucose pyrophosphorylase



Gene: rffH Accession Numbers: EG11454 (EcoCyc), b3789, ECK3781

Synonyms: yifG

Regulation Summary Diagram: ?

Subunit composition of dTDP-glucose pyrophosphorylase = [RffH]4
         dTDP-glucose pyrophosphorylase 2 = RffH

Summary:
dTDP-glucose pyrophosphorylase 2 (RffH) is involved in the synthesis of enterobacterial common antigen (ECA). This enzyme has been shown to have glucose-1-phosphate thymidyltransferase activity.

dTDP-glucose pyrophosphorylase is encoded by the rffH gene which is paralogous to rfbA. The product of rfbA, RmlA, is another dTDP-glucose pyrophosphorylase which catalyzes the same reaction as RffH, but functions in the rhamnose biosynthesis pathway. There is 68% amino acid sequence identity between RffH and RmlA. [Sivaraman02, Marolda95, Stevenson94]

The crystal structure has been reported in the presence of deoxythimidine (dTTP) and Mg2+. The crystal structure shows a tetrameric enzyme with monomers that have an overall α/β fold with two domains, the nucleotide binding and sugar domain. The active site is located at the domain interface. Two conserved aspartate residues act as ligands to the Mg2+ ion. [Sivaraman02]

Locations: cytosol

Map Position: [3,971,631 -> 3,972,512] (85.6 centisomes)
Length: 882 bp / 293 aa

Molecular Weight of Polypeptide: 32.734 kD (from nucleotide sequence)

Molecular Weight of Multimer: 120.0 kD (experimental) [Sivaraman02]

Isozyme Sequence Similarity:
RfbA: YES

Unification Links: ASAP:ABE-0012379 , EchoBASE:EB1423 , EcoGene:EG11454 , EcoliWiki:b3789 , ModBase:P61887 , OU-Microarray:b3789 , PortEco:rffH , PR:PRO_000023784 , Protein Model Portal:P61887 , RefSeq:NP_418236 , RegulonDB:EG11454 , SMR:P61887 , String:511145.b3789 , UniProt:P61887

Relationship Links: InterPro:IN-FAMILY:IPR005835 , InterPro:IN-FAMILY:IPR005907 , Panther:IN-FAMILY:PTHR22572:SF13 , PDB:Structure:1MC3 , Pfam:IN-FAMILY:PF00483

In Paralogous Gene Group: 264 (5 members)

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0009243 - O antigen biosynthetic process Inferred from experiment [Marolda95]
GO:0045226 - extracellular polysaccharide biosynthetic process Inferred from experiment Inferred by computational analysis [GOA01a, Marolda95]
GO:0000271 - polysaccharide biosynthetic process Inferred by computational analysis [UniProtGOA11a]
GO:0009058 - biosynthetic process Inferred by computational analysis [GOA01a]
GO:0009246 - enterobacterial common antigen biosynthetic process Inferred by computational analysis [UniProtGOA12]
Molecular Function: GO:0000287 - magnesium ion binding Inferred from experiment [Sivaraman02]
GO:0008879 - glucose-1-phosphate thymidylyltransferase activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, Marolda95]
GO:0042802 - identical protein binding Inferred from experiment [Sivaraman02]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016779 - nucleotidyltransferase activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]

MultiFun Terms: cell structure surface antigens (ECA, O antigen of LPS)
metabolism biosynthesis of macromolecules (cellular constituents) enterobacterial common antigen (surface glycolipid)
metabolism biosynthesis of macromolecules (cellular constituents) lipopolysaccharide O antigen

Essentiality data for rffH 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 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Credits:
Created 10-Jul-2013 by Kubo A , SRI International
Last-Curated ? 10-Jul-2013 by Kubo A , SRI International


Enzymatic reaction of: dTDP-glucose pyrophosphorylase

Synonyms: glucose-1-phosphate thymidylyltransferase, dTDP-glucose synthase

EC Number: 2.7.7.24

α-D-glucose 1-phosphate + dTTP + H+ <=> dTDP-α-D-glucose + diphosphate

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

This reaction is reversible.

In Pathways: O-antigen building blocks biosynthesis (E. coli) , enterobacterial common antigen biosynthesis , dTDP-L-rhamnose biosynthesis I , dTDP-N-acetylthomosamine biosynthesis

Kinetic Parameters:

Substrate
Km (μM)
Citations
α-D-glucose 1-phosphate
34.0
[Zuccotti01, BRENDA14]
dTDP-α-D-glucose
95.0
[Zuccotti01, BRENDA14]
dTTP
20.5
[Zuccotti01, BRENDA14]
diphosphate
154.0
[Zuccotti01, BRENDA14]

pH(opt): 8 [BRENDA14, Zuccotti01]


Sequence Features

Feature Class Location Citations Comment
Sequence-Conflict 5
[Daniels92, UniProt10a]
Alternate sequence: I → M; UniProt: (in Ref. 1; AAA67589);
Sequence-Conflict 68 -> 69
[Daniels92, UniProt10a]
Alternate sequence: GS → VG; UniProt: (in Ref. 1; AAA67589);
Metal-Binding-Site 108
[UniProt10a]
UniProt: Magnesium;
Sequence-Conflict 130 -> 131
[Daniels92, UniProt10a]
Alternate sequence: AT → P; UniProt: (in Ref. 1; AAA67589);
Metal-Binding-Site 223
[UniProt10a]
UniProt: Magnesium;


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b3789 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11454; 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

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

Daniels92: Daniels DL, Plunkett G, Burland V, Blattner FR (1992). "Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes." Science 1992;257(5071);771-8. PMID: 1379743

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

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

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, 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."

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

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

Marolda95: Marolda CL, Valvano MA (1995). "Genetic analysis of the dTDP-rhamnose biosynthesis region of the Escherichia coli VW187 (O7:K1) rfb gene cluster: identification of functional homologs of rfbB and rfbA in the rff cluster and correct location of the rffE gene." J Bacteriol 1995;177(19);5539-46. PMID: 7559340

Sivaraman02: Sivaraman J, Sauve V, Matte A, Cygler M (2002). "Crystal structure of Escherichia coli glucose-1-phosphate thymidylyltransferase (RffH) complexed with dTTP and Mg2+." J Biol Chem 277(46);44214-9. PMID: 12171937

Stevenson94: Stevenson G, Neal B, Liu D, Hobbs M, Packer NH, Batley M, Redmond JW, Lindquist L, Reeves P (1994). "Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster." J Bacteriol 1994;176(13);4144-56. PMID: 7517391

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 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."

Zuccotti01: Zuccotti S, Zanardi D, Rosano C, Sturla L, Tonetti M, Bolognesi M (2001). "Kinetic and crystallographic analyses support a sequential-ordered bi bi catalytic mechanism for Escherichia coli glucose-1-phosphate thymidylyltransferase." J Mol Biol 313(4);831-43. PMID: 11697907

Other References Related to Gene Regulation

Huerta03: Huerta AM, Collado-Vides J (2003). "Sigma70 promoters in Escherichia coli: specific transcription in dense regions of overlapping promoter-like signals." J Mol Biol 333(2);261-78. PMID: 14529615

Partridge09: Partridge JD, Bodenmiller DM, Humphrys MS, Spiro S (2009). "NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility." Mol Microbiol 73(4);680-94. PMID: 19656291


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 Sun Nov 23, 2014, BIOCYC13A.