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MetaCyc Enzyme: 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Gene: kdsA Accession Numbers: EG10518 (MetaCyc), b1215, ECK1203

Species: Escherichia coli K-12 substr. MG1655

Subunit composition of 3-deoxy-D-manno-octulosonate 8-phosphate synthase = [KdsA]4
         3-deoxy-D-manno-octulosonate 8-phosphate synthase = KdsA

Summary:
3-deoxy-D-manno-octulosonate 8-phosphate (KDO-8-P) synthase is a key enzyme in lipopolysaccharide biosynthesis. It catalyzes the condensation of arabinose-5-phosphate and PEP by cleavage of the C-O bond in PEP, forming the eight-carbon skeleton of 3-deoxy-D-manno-octulosonate, which serves as a linker between the hydrophobic portion of lipopolysaccharide, lipid A, and the hydrophilic polysaccharide chain.

The reaction mechanism has been studied extensively [Baasov93, Dotson93, Dotson95, Liang98, Howe03, Furdui05, Li05].

Various crystal structures of KdsA have been solved [Radaev00, Radaev00a, Wagner00b, Asojo01, Vainer05]. The enzyme appears to be a homotetramer [Radaev00, Radaev00a], although initial biochemical data of the E. coli B enzyme seemed to favor a trimeric form [Ray80]. The crystal structures allowed confirmation of the observed stereochemical preferences and reaction mechanism of the enzyme and provide an explanation for the irreversible nature of the reaction [Vainer05].

Two cysteine residues, C38 and C166, are important for catalytic activity [Salleh96]. Two conserved histidine residues, H97 and H241, are important for catalytic activity, and a third residue, H202, is essential [Sheflyan99]. The wild type E. coli enzyme does not require a metal cofactor, but introduction of a cysteine residue (N26C) that is conserved in metal-requiring enzymes of the same family leads to a lower kcat compared to wild type which increases in the presence of Mn2+ [Oliynyk04, Shulami04].

kdsA expression is highest in early log phase and decreases to a low level in late log and stationary phase [Strohmaier95]. kdsA is an essential gene [Baba06], and temperature-sensitive kdsA mutants stop dividing at the restrictive temperature [Fujishima02].

Citations: [Woisetschlager86]

Locations: cytosol

Map Position: [1,267,388 -> 1,268,242]

Molecular Weight of Polypeptide: 30.833 kD (from nucleotide sequence), 30 kD (experimental) [Dotson95 ]

Molecular Weight of Multimer: 90 kD (experimental) [Ray80]

pI: 6.74

Unification Links: ASAP:ABE-0004077 , CGSC:17671 , DIP:DIP-35940N , EchoBASE:EB0513 , EcoGene:EG10518 , EcoliWiki:b1215 , Mint:MINT-1220797 , ModBase:P0A715 , OU-Microarray:b1215 , PortEco:kdsA , PR:PRO_000023054 , Pride:P0A715 , Protein Model Portal:P0A715 , RefSeq:NP_415733 , RegulonDB:EG10518 , SMR:P0A715 , String:511145.b1215 , UniProt:P0A715

Relationship Links: InterPro:IN-FAMILY:IPR006218 , InterPro:IN-FAMILY:IPR006269 , InterPro:IN-FAMILY:IPR013785 , Panther:IN-FAMILY:PTHR21057:SF2 , PDB:Structure:1D9E , PDB:Structure:1G7U , PDB:Structure:1G7V , PDB:Structure:1GG0 , PDB:Structure:1PHQ , PDB:Structure:1PHW , PDB:Structure:1PL9 , PDB:Structure:1Q3N , PDB:Structure:1X6U , PDB:Structure:1X8F , Pfam:IN-FAMILY:PF00793

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0009244 - lipopolysaccharide core region biosynthetic process Inferred from experiment
GO:0019294 - keto-3-deoxy-D-manno-octulosonic acid biosynthetic process Inferred from experiment Inferred by computational analysis [GOA06]
GO:0008152 - metabolic process Inferred by computational analysis [GOA01a]
GO:0009058 - biosynthetic process Inferred by computational analysis [GOA01a]
GO:0009103 - lipopolysaccharide biosynthetic process Inferred by computational analysis [UniProtGOA12, UniProtGOA11a]
Molecular Function: GO:0008676 - 3-deoxy-8-phosphooctulonate synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, Baasov93]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05, Lasserre06]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA06, GOA01a]

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

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International


Enzymatic reaction of: 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Synonyms: 2-dehydro-3-deoxyphosphooctonate aldolase, phospho-2-dehydro-3-deoxyoctonate aldolase, phospho-2-keto-3-deoxyoctonate aldolase, 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase, KDO 8-phosphate synthase, 2-dehydro-3-deoxy-D-octonate-8-phosphate D-arabinose-5-phosphate-lyase (pyruvate-phosphorylating)

EC Number: 2.5.1.55

D-arabinose 5-phosphate + phosphoenolpyruvate + H2O <=> 3-deoxy-D-manno-octulosonate 8-phosphate + phosphate

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 irreversible in the direction shown.

Alternative Substrates for D-arabinose 5-phosphate [Comment 1 ]: 2-deoxy-D-ribose 5-phosphate [Howe03 ]

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

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Summary:
Initial purification and characterization of the enzyme was from E. coli B [Ray80, Kohen92] and an unnamed E. coli strain [Hedstrom88].

Enzymatic activity is competitively inhibited by the product, 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) with a Ki of 590 µM [Baasov93].

Inhibitors (Competitive): D-ribose 5-phosphate [Ray80, Comment 2] , 3-deoxy-D-manno-octulosonate 8-phosphate [Baasov93]

Inhibitors (Noncompetitive): phosphate [Sheflyan99]

Inhibitors (Unknown Mechanism): diethylpyrocarbonate [Sheflyan99, Ray80, Comment 3] , Cd2+ [Ray80] , Cu2+ [Ray80] , Zn2+ [Ray80] , Hg2+ [Ray80, Comment 4]

Primary Physiological Regulators of Enzyme Activity: D-ribose 5-phosphate

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
phosphoenolpyruvate
6.5
[Salleh96]
phosphoenolpyruvate
4.43
5.31
[Krosky02, BRENDA14]
phosphoenolpyruvate
6.0
6.1
[Shulami04, BRENDA14]
phosphoenolpyruvate
100.0
[Li05, BRENDA14]
D-arabinose 5-phosphate
7.73
[Krosky02, BRENDA14]
D-arabinose 5-phosphate
20.0
[Shulami04, BRENDA14]
D-arabinose 5-phosphate
26.0
[Kohen92, BRENDA14]
D-arabinose 5-phosphate
9.1
[Salleh96]
D-arabinose 5-phosphate
19.0
6.8
[Howe03, BRENDA14]

T(opt): 45 °C [BRENDA14, Ray80]

pH(opt): 4 [BRENDA14, Ray80], 9 [BRENDA14, Ray80]


Sequence Features

Feature Class Location Citations Comment
Sequence-Conflict 22
[Woisetschlager87, UniProt10a]
Alternate sequence: L; UniProt: (in Ref. 1; CAA29067);
Acetylation-Modification 60
[Yu08]
 

History:
10/20/97 Gene b1215 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10518; confirmed by SwissProt match.


References

Asojo01: Asojo O, Friedman J, Adir N, Belakhov V, Shoham Y, Baasov T (2001). "Crystal structures of KDOP synthase in its binary complexes with the substrate phosphoenolpyruvate and with a mechanism-based inhibitor." Biochemistry 40(21);6326-34. PMID: 11371194

Baasov93: Baasov T, Sheffer-Dee-Noor S, Kohen A, Jakob A, Belakhov V (1993). "Catalytic mechanism of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase. The use of synthetic analogues to probe the structure of the putative reaction intermediate." Eur J Biochem 217(3);991-9. PMID: 8223657

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.

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

Dotson93: Dotson GD, Nanjappan P, Reily MD, Woodard RW (1993). "Stereochemistry of 3-deoxyoctulosonate 8-phosphate synthase." Biochemistry 32(46);12392-7. PMID: 8241128

Dotson95: Dotson GD, Dua RK, Clemens JC, Wooten EW, Woodard RW (1995). "Overproduction and one-step purification of Escherichia coli 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase and oxygen transfer studies during catalysis using isotopic-shifted heteronuclear NMR." J Biol Chem 270(23);13698-705. PMID: 7775423

Fujishima02: Fujishima H, Nishimura A, Wachi M, Takagi H, Hirasawa T, Teraoka H, Nishimori K, Kawabata T, Nishikawa K, Nagai K (2002). "kdsA mutations affect FtsZ-ring formation in Escherichia coli K-12." Microbiology 148(Pt 1);103-12. PMID: 11782503

Furdui05: Furdui CM, Sau AK, Yaniv O, Belakhov V, Woodard RW, Baasov T, Anderson KS (2005). "The use of (E)- and (Z)-phosphoenol-3-fluoropyruvate as mechanistic probes reveals significant differences between the active sites of KDO8P and DAHP synthases." Biochemistry 44(19);7326-35. PMID: 15882071

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."

Hedstrom88: Hedstrom L, Abeles R (1988). "3-Deoxy-D-manno-octulosonate-8-phosphate synthase catalyzes the C-O bond cleavage of phosphoenolpyruvate." Biochem Biophys Res Commun 157(2);816-20. PMID: 2904815

Howe03: Howe DL, Sundaram AK, Wu J, Gatti DL, Woodard RW (2003). "Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues." Biochemistry 42(17);4843-54. PMID: 12718525

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

Kohen92: Kohen A, Jakob A, Baasov T (1992). "Mechanistic studies of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase from Escherichia coli." Eur J Biochem 208(2);443-9. PMID: 1521535

Krosky02: Krosky DJ, Alm R, Berg M, Carmel G, Tummino PJ, Xu B, Yang W (2002). "Helicobacter pylori 3-deoxy-D-manno-octulosonate-8-phosphate (KDO-8-P) synthase is a zinc-metalloenzyme." Biochim Biophys Acta 1594(2);297-306. PMID: 11904225

Lasserre06: Lasserre JP, Beyne E, Pyndiah S, Lapaillerie D, Claverol S, Bonneu M (2006). "A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis." Electrophoresis 27(16);3306-21. PMID: 16858726

Li05: Li Z, Sau AK, Furdui CM, Anderson KS (2005). "Probing the role of tightly bound phosphoenolpyruvate in Escherichia coli 3-deoxy-d-manno-octulosonate 8-phosphate synthase catalysis using quantitative time-resolved electrospray ionization mass spectrometry in the millisecond time range." Anal Biochem 343(1);35-47. PMID: 15979047

Liang98: Liang PH, Lewis J, Anderson KS, Kohen A, D'Souza FW, Benenson Y, Baasov T (1998). "Catalytic mechanism of Kdo8P synthase: transient kinetic studies and evaluation of a putative reaction intermediate." Biochemistry 37(46);16390-9. PMID: 9819231

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

Oliynyk04: Oliynyk Z, Briseno-Roa L, Janowitz T, Sondergeld P, Fersht AR (2004). "Designing a metal-binding site in the scaffold of Escherichia coli KDO8PS." Protein Eng Des Sel 17(4);383-90. PMID: 15166313

Radaev00: Radaev S, Dastidar P, Patel M, Woodard RW, Gatti DL (2000). "Structure and mechanism of 3-deoxy-D-manno-octulosonate 8-phosphate synthase." J Biol Chem 2000;275(13);9476-84. PMID: 10734095

Radaev00a: Radaev S, Dastidar P, Patel M, Woodard RW, Gatti DL (2000). "Preliminary X-ray analysis of a new crystal form of the Escherichia coli KDO8P synthase." Acta Crystallogr D Biol Crystallogr 2000;56 ( Pt 4);516-9. PMID: 10739938

Ray80: Ray PH (1980). "Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli." J Bacteriol 1980;141(2);635-44. PMID: 6988389

Salleh96: Salleh HM, Patel MA, Woodard RW (1996). "Essential cysteines in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase from Escherichia coli: analysis by chemical modification and site-directed mutagenesis." Biochemistry 35(27);8942-7. PMID: 8688430

Sheflyan99: Sheflyan GY, Duewel HS, Chen G, Woodard RW (1999). "Identification of essential histidine residues in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase: analysis by chemical modification with diethyl pyrocarbonate and site-directed mutagenesis." Biochemistry 1999;38(43);14320-9. PMID: 10572007

Shulami04: Shulami S, Furdui C, Adir N, Shoham Y, Anderson KS, Baasov T (2004). "A reciprocal single mutation affects the metal requirement of 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthases from Aquifex pyrophilus and Escherichia coli." J Biol Chem 279(43);45110-20. PMID: 15308670

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

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

Vainer05: Vainer R, Belakhov V, Rabkin E, Baasov T, Adir N (2005). "Crystal structures of Escherichia coli KDO8P synthase complexes reveal the source of catalytic irreversibility." J Mol Biol 351(3);641-52. PMID: 16023668

Wagner00b: Wagner T, Kretsinger RH, Bauerle R, Tolbert WD (2000). "3-Deoxy-D-manno-octulosonate-8-phosphate synthase from Escherichia coli. Model of binding of phosphoenolpyruvate and D-arabinose-5-phosphate." J Mol Biol 301(2);233-8. PMID: 10926505

Woisetschlager86: Woisetschlager M, Hogenauer G (1986). "Cloning and characterization of the gene encoding 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli." J Bacteriol 168(1);437-9. PMID: 3531183

Woisetschlager87: Woisetschlager M, Hogenauer G (1987). "The kdsA gene coding for 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase is part of an operon in Escherichia coli." Mol Gen Genet 207(2-3);369-73. PMID: 3039295

Yu08: Yu BJ, Kim JA, Moon JH, Ryu SE, Pan JG (2008). "The diversity of lysine-acetylated proteins in Escherichia coli." J Microbiol Biotechnol 18(9);1529-36. PMID: 18852508


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 18.5 on Sat Dec 20, 2014, biocyc11.