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Escherichia coli K-12 substr. MG1655 Enzyme: 3-deoxy-D-manno-octulosonate cytidylyltransferase



Gene: kdsB Accession Numbers: EG10519 (EcoCyc), b0918, ECK0909

Regulation Summary Diagram: ?

Summary:
CMP-KDO synthetase activates the 8-carbon sugar KDO for incorporation into lipopolysaccharide.

The β-pyranose form of KDO is the preferred substrate of CMP-KDO synthetase [Kohlbrenner85]. A nucleophilic displacement mechanism was proposed for the enzyme [Kohlbrenner87]. Based on kinetic, modelling and EPR studies and the crystal structure of the ternary complex, a reaction mechanism involving two Mg2+ ions was proposed [Heyes09].

A crystal structure of the enzyme has been solved at 2.6 Å resolution [Badger05]; the structure of the ternary complex of KdsB with CTP and 2β-deoxy KDO was solved at 1.9 Å resolution [Heyes09].

Expression of kdsB is growth regulated: it is highest in early log phase and decreases in stationary phase [Strohmaier95]. kdsB is an essential gene [Baba06].

Locations: cytosol

Map Position: [970,075 -> 970,821] (20.91 centisomes)
Length: 747 bp / 248 aa

Molecular Weight of Polypeptide: 27.614 kD (from nucleotide sequence), 32 kD (experimental) [Goldman85 ]

pI: 5.35

Unification Links: ASAP:ABE-0003122 , CGSC:18202 , DIP:DIP-10066N , EchoBASE:EB0514 , EcoGene:EG10519 , EcoliWiki:b0918 , Mint:MINT-1224838 , ModBase:P04951 , OU-Microarray:b0918 , PortEco:kdsB , PR:PRO_000023055 , Pride:P04951 , Protein Model Portal:P04951 , RefSeq:NP_415438 , RegulonDB:EG10519 , SMR:P04951 , String:511145.b0918 , UniProt:P04951

Relationship Links: InterPro:IN-FAMILY:IPR003329 , InterPro:IN-FAMILY:IPR004528 , Panther:IN-FAMILY:PTHR21485:SF4 , PDB:Structure:1VH1 , PDB:Structure:3K8D , PDB:Structure:3K8E , Pfam:IN-FAMILY:PF02348

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0019294 - keto-3-deoxy-D-manno-octulosonic acid biosynthetic process Inferred from experiment Inferred by computational analysis [GOA06, Goldman85]
GO:0009103 - lipopolysaccharide biosynthetic process Inferred by computational analysis [UniProtGOA12, UniProtGOA11a, GOA01a]
GO:0033468 - CMP-keto-3-deoxy-D-manno-octulosonic acid biosynthetic process Inferred by computational analysis [UniProtGOA12]
Molecular Function: GO:0000287 - magnesium ion binding Inferred from experiment [Heyes09]
GO:0005515 - protein binding Inferred from experiment [Rajagopala14, Butland05]
GO:0008690 - 3-deoxy-manno-octulosonate cytidylyltransferase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, Goldman85]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016779 - nucleotidyltransferase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA06, GOA01a]

MultiFun Terms: metabolism biosynthesis of macromolecules (cellular constituents) lipopolysaccharide core region

Essentiality data for kdsB 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 ? 09-Nov-2011 by Keseler I , SRI International


Enzymatic reaction of: 3-deoxy-D-manno-octulosonate cytidylyltransferase

Synonyms: CPM-3-deoxy-D-manno-octulosonate pyrophosphorylase, CTP:3-deoxy-D-manno-octulosonate cytidylyltransferase, CTP:CMP-3-deoxy-D-manno-octulosonate-cytidylyl-transferase, CMP-KDO synthetase

EC Number: 2.7.7.38

3-deoxy-D-manno-octulosonate + CTP <=> CMP-3-deoxy-D-manno-octulosonate + diphosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

Alternative Substrates [Comment 2]:

In Pathways: superpathway of (KDO)2-lipid A biosynthesis , superpathway of lipopolysaccharide biosynthesis , CMP-KDO biosynthesis I

Summary:
The enzyme was first purified from E. coli O111-B4 [Ghalambor66] and E. coli B [Ray81].

2β-deoxy KDO is a competitive inhibitor with respect to KDO with a Ki of 2.03 µM and is a non-competitive inhibitor with respect to CTP [Heyes09].

Cofactors or Prosthetic Groups: Mg2+ [Heyes09]

Inhibitors (Competitive): 2β-deoxy Kdo [Heyes09] , CMP [Heyes09]

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
3-deoxy-D-manno-octulosonate
290.0
[Ray81, BRENDA14]
3-deoxy-D-manno-octulosonate
390.0
[Ray82, BRENDA14]
3-deoxy-D-manno-octulosonate
800.0
[Ghalambor66, BRENDA14]
3-deoxy-D-manno-octulosonate
2000.0
[Rosenow95, BRENDA14]
3-deoxy-D-manno-octulosonate
97.9
[Heyes09]
3-deoxy-D-manno-octulosonate
280.0, 520.0
12.5, 21.9
[Yi11, BRENDA14]
CTP
200.0
[Ray82, BRENDA14]
CTP
220.0
[Ghalambor66, BRENDA14]
CTP
330.0, 590.0
[Yi11, BRENDA14]
CTP
2500.0
[Rosenow95, BRENDA14]
CTP
4.8
[Heyes09]

pH(opt): 7.8 [BRENDA14, Ghalambor66], 9.3 [BRENDA14, Yi11], 9.5 [BRENDA14, Rosenow95], 9.6 [BRENDA14, Ray81]


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Goldman86]
 
Chain 2 -> 248
[UniProt09]
UniProt: 3-deoxy-manno-octulosonate cytidylyltransferase;


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b0918 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10519; 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

Badger05: Badger J, Sauder JM, Adams JM, Antonysamy S, Bain K, Bergseid MG, Buchanan SG, Buchanan MD, Batiyenko Y, Christopher JA, Emtage S, Eroshkina A, Feil I, Furlong EB, Gajiwala KS, Gao X, He D, Hendle J, Huber A, Hoda K, Kearins P, Kissinger C, Laubert B, Lewis HA, Lin J, Loomis K, Lorimer D, Louie G, Maletic M, Marsh CD, Miller I, Molinari J, Muller-Dieckmann HJ, Newman JM, Noland BW, Pagarigan B, Park F, Peat TS, Post KW, Radojicic S, Ramos A, Romero R, Rutter ME, Sanderson WE, Schwinn KD, Tresser J, Winhoven J, Wright TA, Wu L, Xu J, Harris TJ (2005). "Structural analysis of a set of proteins resulting from a bacterial genomics project." Proteins 60(4);787-96. PMID: 16021622

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

Butland05: Butland G, Peregrin-Alvarez JM, Li J, Yang W, Yang X, Canadien V, Starostine A, Richards D, Beattie B, Krogan N, Davey M, Parkinson J, Greenblatt J, Emili A (2005). "Interaction network containing conserved and essential protein complexes in Escherichia coli." Nature 433(7025);531-7. PMID: 15690043

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

Ghalambor66: Ghalambor MA, Heath EC (1966). "The biosynthesis of cell wall lipopolysaccharide in Escherichia coli. IV. Purification and properties of cytidine monophosphate 3-deoxy-d-manno-octulosonate synthetase." J Biol Chem 1966;241(13);3216-21. PMID: 5330266

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."

Goldman85: Goldman RC, Kohlbrenner WE (1985). "Molecular cloning of the structural gene coding for CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase from Escherichia coli K-12." J Bacteriol 163(1);256-61. PMID: 2989246

Goldman86: Goldman RC, Bolling TJ, Kohlbrenner WE, Kim Y, Fox JL (1986). "Primary structure of CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from Escherichia coli." J Biol Chem 1986;261(34);15831-5. PMID: 3023327

Heyes09: Heyes DJ, Levy C, Lafite P, Roberts IS, Goldrick M, Stachulski AV, Rossington SB, Stanford D, Rigby SE, Scrutton NS, Leys D (2009). "Structure-based mechanism of CMP-2-keto-3-deoxymanno-octulonic acid synthetase: convergent evolution of a sugar-activating enzyme with DNA/RNA polymerases." J Biol Chem 284(51);35514-23. PMID: 19815542

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

Kohlbrenner85: Kohlbrenner WE, Fesik SW (1985). "Determination of the anomeric specificity of the Escherichia coli CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase by 13C NMR spectroscopy." J Biol Chem 260(27);14695-700. PMID: 2997221

Kohlbrenner87: Kohlbrenner WE, Nuss MM, Fesik SW (1987). "31P and 13C NMR studies of oxygen transfer during catalysis by 3-deoxy-D-manno-octulosonate cytidylyltransferase from Escherichia coli." J Biol Chem 262(10);4534-7. PMID: 3031027

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

Ray81: Ray PH, Benedict CD, Grasmuk H (1981). "Purification and characterization of cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase." J Bacteriol 1981;145(3);1273-80. PMID: 6259128

Ray82: Ray PH, Benedict CD (1982). "CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase)." Methods Enzymol 83;535-40. PMID: 6285137

Rosenow95: Rosenow C, Roberts IS, Jann K (1995). "Isolation from recombinant Escherichia coli and characterization of CMP-Kdo synthetase, involved in the expression of the capsular K5 polysaccharide (K-CKS)." FEMS Microbiol Lett 125(2-3);159-64. PMID: 7875563

Strohmaier95: Strohmaier H, Remler P, Renner W, Hogenauer G (1995). "Expression of genes kdsA and kdsB involved in 3-deoxy-D-manno-octulosonic acid metabolism and biosynthesis of enterobacterial lipopolysaccharide is growth phase regulated primarily at the transcriptional level in Escherichia coli K-12." J Bacteriol 177(15);4488-500. PMID: 7543480

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

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

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."

Yi11: Yi L, Velasquez MS, Holler TP, Woodard RW (2011). "A simple assay for 3-deoxy-d-manno-octulosonate cytidylyltransferase and its use as a pathway screen." Anal Biochem 416(2);152-8. PMID: 21669179


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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 Thu Dec 25, 2014, BIOCYC13B.