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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: ?

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].

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].

Reviews: [Raetz02, Heinrichs98, Schnaitman93]

Citations: [Roncero92, Brabetz97, Gronow09]

Gene Citations: [Klena92, Sirisena94]

Locations: cytosol, inner membrane

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

Molecular Weight of Polypeptide: 35.544 kD (from nucleotide sequence), 36 kD (experimental) [Chen93d ]

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: ?

GO Terms:

Biological Process: GO:0009244 - lipopolysaccharide core region biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, Chen93d, 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: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:
Last-Curated ? 02-Dec-2011 by Keseler I , SRI International


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

EC Number: 2.4.-.-

(KDO)2-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
(KDO)2-lipid A
9.0
4.7
[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): ?

Transcription Units:

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

Chen93d: 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

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

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

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

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

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

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

Raina91: 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


Report Errors or Provide Feedback
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 Fri Dec 19, 2014, BIOCYC14A.