MetaCyc Enzyme: lipopolysaccharide core heptose (I) kinase

Gene: waaP Accession Numbers: EG11340 (MetaCyc), b3630, ECK3620

Synonyms: rfaP

Species: Escherichia coli K-12 substr. MG1655

The lipopolysaccharide of E. coli K-12 consists of two major components: the hydrophobic lipid A moiety inserted into the outer membrane and the phosphorylated core oligosaccharide [Raetz02]. E. coli K-12 does not produce O antigen to attach to the LPS core due to a defect in the rfb gene cluster which can be complemented with genes from a second, independent rfb mutant to produce an O16 type O antigen [Stevenson94]. E. coli K-12 may have two major pathways for LPS biosynthesis. One generates LPS cores suitable for O antigen attachment, and a second generates lipooligosaccharides (LOS) with modifications to the core structure which prevent O antigen attachment [Klena92a].

The WaaP protein is a lipopolysaccharide (LPS) kinase; it is required for the addition of phosphate to O-4 of the heptose I residue in the lipopolysaccharide core [Parker92, Yethon98, Yethon01]. This modification appears to be critical for the stability of the outer membrane.

Assays using WaaP from E. coli strain F470 reveal that Mg2+ is required for WaaP activity, and the optimum pH is between 8.0 and 9.0 [Yethon01]. Also, WaaP activity is partially dependent upon an intact waaG in order to phosphorylate the HepI residue in F470 [Yethon00].

waaP+ reversed the deep rough phenotype of a waaGPBO deletion mutant [Parker92]. Mutation of the waaP gene causes hypersensitivity to novobiocin and sodium dodecyl sulfate (and generally, hydrophobic antibiotics and detergents) but these mutants do not show altered outer membrane protein profiles [Yethon98, Yethon00].

A transposon insertion in waaP resulted in production of less Type I fimbriae as compared with the W3110 parental strain [Genevaux99]. Inactivation of waaP can suppress the lethality of a ΔseqA/cold-sensitive recA double mutant [Rotman09].

Recombiant N-terminally His-tagged WaaP was co-expressed with E. coli chaperones GroES and GroEL which improved solubility and allowed purification, although the majority of protein remained in inclusion bodies. There was also evidence for membrane association. Homology analysis of WaaP revealed greater than 80% identity and 90% similarity between E. coli and Salmonella enterica, and 55% identity and 70% similarity between these organisms and Pseudomonas aeruginosa. E. coli residues 159-171 were absolutely conserved. Only 10-15% identity and 25-30% similarity was seen between E. coli WaaP and eukaryotic protein kinases, with conservation of residues known to be catalytically important. A waaP site-directed mutant D162A lost kinase activity showing that this is an essential residue [Yethon01].

The chromosomal waa region (formerly rfa) contains the major core-oligosaccharide assembly operons in E. coli [Raetz02, Raetz07]. The current nomenclature system was proposed originally in [Reeves96] and [Heinrichs98] and followed thereafter.

Reviews: [Raetz02, Heinrichs98, Schnaitman93]

Locations: membrane

Map Position: [3,803,176 <- 3,803,973]

Molecular Weight of Polypeptide: 30.872 kD (from nucleotide sequence), 33.0 kD (experimental) [Yethon01 ]

Unification Links: ASAP:ABE-0011866 , CGSC:298 , EchoBASE:EB1316 , EcoGene:EG11340 , Ecol199310Cyc:C4454 , EcoliWiki:b3630 , Entrez-gene:948150 , OU-Microarray:b3630 , PortEco:rfaP , PR:PRO_000023724 , Pride:P25741 , Protein Model Portal:P25741 , RefSeq:NP_418087 , RegulonDB:EG11340 , String:511145.b3630 , UniProt:P25741

Relationship Links: InterPro:IN-FAMILY:IPR010440 , InterPro:IN-FAMILY:IPR011009 , InterPro:IN-FAMILY:IPR017172 , Pfam:IN-FAMILY:PF06293

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0009244 - lipopolysaccharide core region biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, Parker92]
GO:0009103 - lipopolysaccharide biosynthetic process Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0016310 - phosphorylation Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0016301 - kinase activity Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA01, Yethon01]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
GO:0016772 - transferase activity, transferring phosphorus-containing groups Inferred by computational analysis [GOA01]
GO:0016773 - phosphotransferase activity, alcohol group as acceptor Inferred by computational analysis [GOA01]
Cellular Component: GO:0016020 - membrane Inferred by computational analysis [GOA01]

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

Imported from EcoCyc 02-Jun-2015 by Paley S , SRI International

Enzymatic reaction of: lipopolysaccharide core heptose (I) kinase

EC Number: 2.7.1.-

glucosyl-(heptosyl)2-Kdo2-lipid A + ATP <=> glucosyl-(heptosyl)2-Kdo2-lipid A-phosphate + ADP + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is favored in the direction shown.

In Pathways: superpathway of lipopolysaccharide biosynthesis , Lipid A-core biosynthesis

Imported from EcoCyc 02-Jun-2015 by Paley S , SRI International

Lipopolysaccharide (LPS) was used as the acceptor in the in vitro assay. Mg2+ was required for activity and Ca2+ could not substitute [Yethon01].

Cofactors or Prosthetic Groups: Mg2+ [Yethon01]

Kinetic Parameters:

Km (μM)
Vmax (µmol mg-1 min-1)
glucosyl-(heptosyl)2-Kdo2-lipid A

pH(opt): 8-9 [Yethon01]

Sequence Features

Feature Class Location Citations Comment
Active-Site 162
UniProt: Non-Experimental Qualifier: potential;

10/20/97 Gene b3630 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11340; confirmed by SwissProt match.


Genevaux99: Genevaux P, Bauda P, DuBow MS, Oudega B (1999). "Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion." Arch Microbiol 172(1);1-8. PMID: 10398745

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

Heinrichs98: Heinrichs DE, Yethon JA, Whitfield C (1998). "Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica." Mol Microbiol 30(2);221-32. PMID: 9791168

Klena92a: Klena JD, Ashford RS, Schnaitman CA (1992). "Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen." J Bacteriol 174(22);7297-307. PMID: 1385388

Parker92: Parker CT, Kloser AW, Schnaitman CA, Stein MA, Gottesman S, Gibson BW (1992). "Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12." J Bacteriol 174(8);2525-38. PMID: 1348243

Raetz02: Raetz CR, Whitfield C (2002). "Lipopolysaccharide endotoxins." Annu Rev Biochem 71;635-700. PMID: 12045108

Raetz07: Raetz CR, Reynolds CM, Trent MS, Bishop RE (2007). "Lipid A modification systems in gram-negative bacteria." Annu Rev Biochem 76;295-329. PMID: 17362200

Reeves96: Reeves PR, Hobbs M, Valvano MA, Skurnik M, Whitfield C, Coplin D, Kido N, Klena J, Maskell D, Raetz CR, Rick PD (1996). "Bacterial polysaccharide synthesis and gene nomenclature." Trends Microbiol 4(12);495-503. PMID: 9004408

Rotman09: Rotman E, Bratcher P, Kuzminov A (2009). "Reduced lipopolysaccharide phosphorylation in Escherichia coli lowers the elevated ori/ter ratio in seqA mutants." Mol Microbiol 72(5);1273-92. PMID: 19432803

Schnaitman93: Schnaitman CA, Klena JD (1993). "Genetics of lipopolysaccharide biosynthesis in enteric bacteria." Microbiol Rev 57(3);655-82. PMID: 7504166

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 2010-07 released on 2010-06-15 00:00:00." Database.

UniProtGOA11: 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."

Yethon00: Yethon JA, Vinogradov E, Perry MB, Whitfield C (2000). "Mutation of the lipopolysaccharide core glycosyltransferase encoded by waaG destabilizes the outer membrane of Escherichia coli by interfering with core phosphorylation." J Bacteriol 182(19);5620-3. PMID: 10986272

Yethon01: Yethon JA, Whitfield C (2001). "Purification and characterization of WaaP from Escherichia coli, a lipopolysaccharide kinase essential for outer membrane stability." J Biol Chem 276(8);5498-504. PMID: 11069912

Yethon98: Yethon JA, Heinrichs DE, Monteiro MA, Perry MB, Whitfield C (1998). "Involvement of waaY, waaQ, and waaP in the modification of Escherichia coli lipopolysaccharide and their role in the formation of a stable outer membrane." J Biol Chem 273(41);26310-6. PMID: 9756860

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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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