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Escherichia coli K-12 substr. MG1655 Enzyme: ADP-heptose:LPS heptosyltransferase I



Gene: waaC Accession Numbers: EG11189 (EcoCyc), b3621, ECK3611

Synonyms: rfa-2, rfaC, yibC

Regulation Summary Diagram: ?

Regulation summary diagram for waaC

Summary:
ADP-heptose:LPS heptosyltransferase I (HepI) is the enzyme responsible for transfer of the first heptose sugar onto the Kdo2 moiety of the lipopolysaccharide inner core [Kadrmas98, Gronow00].

HepI is able to catalyze heptose transfer to underacylated and fully deacylated Kdo2-lipid A analogs; this activity does not require addition of detergent. Thus, the enzyme appears to only recognize the Kdo sugar region of these acceptor molecules [Czyzyk11].

Crystal structures of WaaC from the pathogenic strain RS218 have been solved, providing insight into the catalytic mechanism [Grizot06]. WaaC is considered to be an antimicrobial drug target, and small molecule inhibitors have been identified [Moreau08, Durka12].

A waaC mutant has a defect in the LPS core heptose region [Beher81], causing a deep-rough phenotype; the lipopolysaccharide of the mutant strain is heptoseless and contains 2-keto-3-deoxyoctulosonic acid (Kdo) as its only core sugar [Coleman85]. A Δ(waaC lpxL lpxM lpxP) strain is viable under slow growth conditions in minimal medium at low temperature (23°C) and synthesizes only Kdo2-lipid IVA. Overexpression of msbA partially rescues the growth defect of that strain. Lack of waaC induces the σE extracytoplasmic stress response [Klein09].

Overexpression of waaC leads to formation of biofilms with abnormal architecture [Tenorio03]. In waaC, waaE, waaF and waaG mutants biofilm formation was significantly increased relative to the parental strain [Nakao12].

The waaC gene is located in the first of three operons in the rfa locus [Roncero92]. In E. coli K-12 W3110 inactivation of both chromosomal waaC and the heptosyltransferase waaF by allelic-replacement mutagenesis resulted in Re-LPS with highly 2-aminoethanol phosphate substituted tetrasaccharide [Brabetz97] (Re-LPS is a rough LPS mutant [GarciaVerdugo05]). In E. coli K-12 strains deficient in waaC and waaF heterologously expressing Chlamydial waaA, it was shown that Kdo-(2->8)-Kdo(2->4), but not Kdo-(2->4)-Kdo(2->4) could serve as a heptose acceptor for E. coli WaaC [Gronow09].

In E. coli K-12 W3110 two waaC mutants were shown to be resistant to infection by bacteriophage mEp213 [ReyesCortes12]. Mutant selection on the antibiotic tigecycline included a mutant in waaC with low-level resistance [Linkevicius13]. WaaC was also shown to directly interact with LapB in vivo, the latter being a heat shock protein involved in lipopolysaccharide assembly [Klein14].

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]

Gene Citations: [Klena92, Sirisena94]

Locations: cytosol, inner membrane

Map Position: [3,794,002 -> 3,794,961] (81.77 centisomes, 294°)
Length: 960 bp / 319 aa

Molecular Weight of Polypeptide: 35.544 kD (from nucleotide sequence), 36 kD (experimental) [Chen93a ]

Unification Links: ASAP:ABE-0011847 , CGSC:300 , EchoBASE:EB1175 , EcoGene:EG11189 , EcoliWiki:b3621 , Entrez-gene:948136 , ModBase:P24173 , OU-Microarray:b3621 , PortEco:rfaC , PR:PRO_000023717 , Protein Model Portal:P24173 , RefSeq:NP_418078 , RegulonDB:EG11189 , SMR:P24173 , String:511145.b3621 , UniProt:P24173

Relationship Links: CAZy:IN-FAMILY:GT9 , InterPro:IN-FAMILY:IPR002201 , InterPro:IN-FAMILY:IPR011908 , PDB:Ortholog:2H1H , Pfam:IN-FAMILY:PF01075

In Paralogous Gene Group: 543 (2 members)

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, Chen93a, Beher81, Coleman85]
GO:0008152 - metabolic process Inferred by computational analysis [GOA01]
GO:0009103 - lipopolysaccharide biosynthetic process Inferred by computational analysis [UniProtGOA11, GOA01]
Molecular Function: GO:0005515 - protein binding Inferred from experiment [Klein14]
GO:0008920 - lipopolysaccharide heptosyltransferase activity Inferred from experiment Inferred by computational analysis [GOA01, Gronow00, Czyzyk11, Kadrmas98]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
GO:0016757 - transferase activity, transferring glycosyl groups Inferred by computational analysis [UniProtGOA11, GOA01]
Cellular Component: GO:0005829 - cytosol Inferred by computational analysis [DiazMejia09]
GO:0005886 - plasma membrane

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

Essentiality data for waaC knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 1]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 2]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 1]
Yes [Feist07, Comment 3]

Credits:
Curated 02-Dec-2011 by Keseler I , SRI International
Last-Curated ? 16-Jan-2015 by Fulcher C , SRI International


Enzymatic reaction of: ADP-heptose:Kdo2-lipid A heptosyltransferase (ADP-heptose:LPS heptosyltransferase I)

EC Number: 2.4.-.-

α-D-Kdo-(2→4)-α-D-Kdo-(2→6)-lipid A + ADP-L-glycero-β-D-manno-heptose <=> heptosyl-Kdo2-lipid A + 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

Summary:
Addition of detergent (Triton X-100) increases activity of the enzyme [Czyzyk11].

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
α-D-Kdo-(2→4)-α-D-Kdo-(2→6)-lipid A
9.0
4.7
0.52
[Czyzyk11]


Enzymatic reaction of: ADP-heptose:Kdo2-lipid IVA-heptosyltransferase (ADP-heptose:LPS heptosyltransferase I)

α-Kdo-(2->4)-α-Kdo-(2->6)-lipid IVA + ADP-L-glycero-β-D-manno-heptose <=> heptosyl-Kdo2-lipid IVA + 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.

Reversibility of this reaction is unspecified.

Alternative Substrates for ADP-L-glycero-β-D-manno-heptose: ADP-mannose [Kadrmas96 , Kadrmas98 ]

Summary:
The enzyme activity was characterized using the commercially available substrate ADP-mannose in place of ADP-L-glycero-D-manno-heptose. Addition of detergent (Triton X-100) is required for activity [Kadrmas98]. The physiological substrate was purified and characterized as ADP-L-glycero-β-D-manno-heptopyranose [Gronow01].

Kinetic Parameters:

Substrate
Km (μM)
Citations
α-Kdo-(2->4)-α-Kdo-(2->6)-lipid IVA
4.5
[Kadrmas98]
ADP-mannose
1500.0
[Kadrmas98]

pH(opt): 7.5 [Kadrmas98]


Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Units:

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Notes:

History:
10/20/97 Gene b3621 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11189; 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

Beher81: Beher MG, Schnaitman CA (1981). "Regulation of the OmpA outer membrane protein of Escherichia coli." J Bacteriol 147(3);972-85. PMID: 7024253

Brabetz97: Brabetz W, Muller-Loennies S, Holst O, Brade H (1997). "Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution." Eur J Biochem 247(2);716-24. PMID: 9266718

Chen93a: Chen L, Coleman WG (1993). "Cloning and characterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner core synthesis." J Bacteriol 175(9);2534-40. PMID: 8478319

Coleman85: Coleman WG, Deshpande KS (1985). "New cysE-pyrE-linked rfa mutation in Escherichia coli K-12 that results in a heptoseless lipopolysaccharide." J Bacteriol 161(3);1209-14. PMID: 3882666

Czyzyk11: Czyzyk DJ, Liu C, Taylor EA (2011). "Lipopolysaccharide biosynthesis without the lipids: recognition promiscuity of Escherichia coli heptosyltransferase I." Biochemistry 50(49);10570-2. PMID: 22059588

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

Durka12: Durka M, Buffet K, Iehl J, Holler M, Nierengarten JF, Vincent SP (2012). "The inhibition of liposaccharide heptosyltransferase WaaC with multivalent glycosylated fullerenes: a new mode of glycosyltransferase inhibition." Chemistry 18(2);641-51. PMID: 22147564

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

GarciaVerdugo05: Garcia-Verdugo I, Sanchez-Barbero F, Soldau K, Tobias PS, Casals C (2005). "Interaction of SP-A (surfactant protein A) with bacterial rough lipopolysaccharide (Re-LPS), and effects of SP-A on the binding of Re-LPS to CD14 and LPS-binding protein." Biochem J 391(Pt 1);115-24. PMID: 15932345

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

Grizot06: Grizot S, Salem M, Vongsouthi V, Durand L, Moreau F, Dohi H, Vincent S, Escaich S, Ducruix A (2006). "Structure of the Escherichia coli heptosyltransferase WaaC: binary complexes with ADP and ADP-2-deoxy-2-fluoro heptose." J Mol Biol 363(2);383-94. PMID: 16963083

Gronow00: Gronow S, Brabetz W, Brade H (2000). "Comparative functional characterization in vitro of heptosyltransferase I (WaaC) and II (WaaF) from Escherichia coli." Eur J Biochem 267(22);6602-11. PMID: 11054112

Gronow01: Gronow S, Oertelt C, Ervela E, Zamyatina A, Kosma P, Skurnik M, Holst O (2001). "Characterization of the physiological substrate for lipopolysaccharide heptosyltransferases I and II." J Endotoxin Res 7(4);263-70. PMID: 11717579

Gronow09: Gronow S, Lindner B, Brade H, Muller-Loennies S (2009). "Kdo-(2 --> 8)-Kdo-(2 --> 4)-Kdo but not Kdo-(2 --> 4)-Kdo-(2 --> 4)-Kdo is an acceptor for transfer of L-glycero-alpha-D-manno-heptose by Escherichia coli heptosyltransferase I (WaaC)." Innate Immun 15(1);13-23. PMID: 19201821

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

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

Kadrmas96: Kadrmas JL, Brozek KA, Raetz CR (1996). "Lipopolysaccharide core glycosylation in Rhizobium leguminosarum. An unusual mannosyl transferase resembling the heptosyl transferase I of Escherichia coli." J Biol Chem 271(50);32119-25. PMID: 8943265

Kadrmas98: Kadrmas JL, Raetz CR (1998). "Enzymatic synthesis of lipopolysaccharide in Escherichia coli. Purification and properties of heptosyltransferase i." J Biol Chem 273(5);2799-807. PMID: 9446588

Klein09: Klein G, Lindner B, Brabetz W, Brade H, Raina S (2009). "Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases: minimal lipopolysaccharide structure and induction of envelope stress response." J Biol Chem 284(23);15369-89. PMID: 19346244

Klein14: Klein G, Kobylak N, Lindner B, Stupak A, Raina S (2014). "Assembly of lipopolysaccharide in Escherichia coli requires the essential LapB heat shock protein." J Biol Chem 289(21);14829-53. PMID: 24722986

Klena92: Klena JD, Pradel E, Schnaitman CA (1992). "Comparison of lipopolysaccharide biosynthesis genes rfaK, rfaL, rfaY, and rfaZ of Escherichia coli K-12 and Salmonella typhimurium." J Bacteriol 174(14);4746-52. PMID: 1624462

Linkevicius13: Linkevicius M, Sandegren L, Andersson DI (2013). "Mechanisms and fitness costs of tigecycline resistance in Escherichia coli." J Antimicrob Chemother 68(12);2809-19. PMID: 23843301

Moreau08: Moreau F, Desroy N, Genevard JM, Vongsouthi V, Gerusz V, Le Fralliec G, Oliveira C, Floquet S, Denis A, Escaich S, Wolf K, Busemann M, Aschenbrenner A (2008). "Discovery of new Gram-negative antivirulence drugs: structure and properties of novel E. coli WaaC inhibitors." Bioorg Med Chem Lett 18(14);4022-6. PMID: 18571407

Nakao12: Nakao R, Ramstedt M, Wai SN, Uhlin BE (2012). "Enhanced biofilm formation by Escherichia coli LPS mutants defective in Hep biosynthesis." PLoS One 7(12);e51241. PMID: 23284671

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

ReyesCortes12: Reyes-Cortes R, Martinez-Penafiel E, Martinez-Perez F, de la Garza M, Kameyama L (2012). "A novel strategy to isolate cell-envelope mutants resistant to phage infection: bacteriophage mEp213 requires lipopolysaccharides in addition to FhuA to enter Escherichia coli K-12." Microbiology 158(Pt 12);3063-71. PMID: 23103976

Roncero92: Roncero C, Casadaban MJ (1992). "Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12: three operons in the rfa locus." J Bacteriol 174(10);3250-60. PMID: 1577693

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

Sirisena94: Sirisena DM, MacLachlan PR, Liu SL, Hessel A, Sanderson KE (1994). "Molecular analysis of the rfaD gene, for heptose synthesis, and the rfaF gene, for heptose transfer, in lipopolysaccharide synthesis in Salmonella typhimurium." J Bacteriol 1994;176(8);2379-85. PMID: 8157607

Tenorio03: Tenorio E, Saeki T, Fujita K, Kitakawa M, Baba T, Mori H, Isono K (2003). "Systematic characterization of Escherichia coli genes/ORFs affecting biofilm formation." FEMS Microbiol Lett 225(1);107-14. PMID: 12900028

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."

Other References Related to Gene Regulation

Dartigalongue01: Dartigalongue C, Missiakas D, Raina S (2001). "Characterization of the Escherichia coli sigma E regulon." J Biol Chem 276(24);20866-75. PMID: 11274153

Pegues90: Pegues JC, Chen LS, Gordon AW, Ding L, Coleman WG (1990). "Cloning, expression, and characterization of the Escherichia coli K-12 rfaD gene." J Bacteriol 172(8);4652-60. PMID: 2198271

Raina91a: Raina S, Georgopoulos C (1991). "The htrM gene, whose product is essential for Escherichia coli viability only at elevated temperatures, is identical to the rfaD gene." Nucleic Acids Res 19(14);3811-9. PMID: 1861974


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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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