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Escherichia coli K-12 substr. MG1655 Enzyme: 3-deoxy-D-manno-octulosonate 8-phosphate synthase



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

Regulation Summary Diagram: ?

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, Li05b].

Various crystal structures of KdsA have been solved [Radaev00, Radaev00a, Wagner00a, 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 [Ray80a]. 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] (27.32 centisomes)
Length: 855 bp / 284 aa

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

Molecular Weight of Multimer: 90 kD (experimental) [Ray80a]

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 [GOA01]
GO:0009058 - biosynthetic process Inferred by computational analysis [GOA01]
GO:0009103 - lipopolysaccharide biosynthetic process Inferred by computational analysis [UniProtGOA12, UniProtGOA11]
Molecular Function: GO:0008676 - 3-deoxy-8-phosphooctulonate synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Baasov93]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05, Lasserre06]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11a, UniProtGOA11, GOA06, GOA01]

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

Essentiality data for kdsA 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 No 37 Aerobic 7   No [Baba06, Comment 2]

Credits:
Last-Curated ? 12-Apr-2007 by Keseler I , 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 3 ]: 2-deoxy-D-ribose 5-phosphate [Howe03 ]

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

Summary:
Initial purification and characterization of the enzyme was from E. coli B [Ray80a, 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 [Ray80a, Comment 4] , 3-deoxy-D-manno-octulosonate 8-phosphate [Baasov93]

Inhibitors (Noncompetitive): phosphate [Sheflyan99]

Inhibitors (Unknown Mechanism): diethylpyrocarbonate [Sheflyan99, Ray80a, Comment 5] , Cd2+ [Ray80a] , Cu2+ [Ray80a] , Zn2+ [Ray80a] , Hg2+ [Ray80a, Comment 6]

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
[Li05b, 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, Ray80a]

pH(opt): 4 [BRENDA14, Ray80a], 9 [BRENDA14, Ray80a]


Sequence Features

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


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

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

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, 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."

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

Li05b: 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

Ray80a: 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 manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms 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

Wagner00a: 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


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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 18, 2014, BIOCYC13B.