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Escherichia coli K-12 substr. MG1655 Enzyme: ADP-L-glycero-D-mannoheptose-6-epimerase



Gene: rfaD Accession Numbers: EG10838 (EcoCyc), b3619, ECK3609

Synonyms: waaD, hldD, htrM, nbsB

Regulation Summary Diagram: ?

Subunit composition of ADP-L-glycero-D-mannoheptose-6-epimerase = [RfaD]5
         ADP-L-glycero-D-mannoheptose-6-epimerase = RfaD

Summary:
The rfaD gene encodes ADP-L-glycero-D-mannoheptose-6-epimerase, the last enzyme in the pathway for synthesis of the ADP-heptose precursor of core LPS [Kneidinger02]. The enzyme is glycosylated [Ding94]. It was initially thought to contain NAD+ as a cofactor [Ding94], but the cofactor found in the crystal structure was NADP+ [Deacon00]. Subsequent studies showed that the enzyme has a preference of NADP+ over NAD+ [Ni01].

A crystal structure of RfaD has been solved at 2 Å resolution [Deacon00]. The enzyme was initially thought to form a homohexamer [Ding94], but appears as a homopentamer in the crystal structure [Deacon00]. A reaction mechanism involving transient oxidation of the C-6'' stereocenter of the substrate and transient reduction of the NADP+ cofactor has been proposed [Read04, Morrison05]. Catalysis involves two basic residues, Tyr140 and Lys178 [Morrison07]. Only very small amounts of the transiently oxidized 6''-keto intermediate are released from the enzyme during catalysis [Mayer07].

An rfaD null mutant strain is unable to grow above 42°C and has a mucoid phenotype [Karow91]. An rfaD mutant is supersensitive to novobiocin and other hydrophobic drugs, and its lipopolysaccharide contains the stereoisomer D-glycero-D-manno-heptose rather than L-glycero-D-manno-heptose [Coleman79, Coleman83]. An rfaD::cat mutation results in induction of mucoidy and increased expression of gabT, which is likely due to increased expression of RpoS [Joloba04].

rfaD expression increases under high temperature conditions and is regulated by the alternative sigma factor σ32 [Karow91, Raina91].

Gene Citations: [Klena92, Sirisena94]

Locations: cytosol, membrane

Map Position: [3,792,010 -> 3,792,942] (81.73 centisomes)
Length: 933 bp / 310 aa

Molecular Weight of Polypeptide: 34.893 kD (from nucleotide sequence), 37 kD (experimental) [Pegues90 ]

Molecular Weight of Multimer: 240 kD (experimental) [Ding94]

Unification Links: ASAP:ABE-0011843 , CGSC:299 , DIP:DIP-35958N , EchoBASE:EB0831 , EcoGene:EG10838 , EcoliWiki:b3619 , ModBase:P67910 , OU-Microarray:b3619 , PortEco:rfaD , PR:PRO_000022904 , Pride:P67910 , Protein Model Portal:P67910 , RefSeq:NP_418076 , RegulonDB:EG10838 , SMR:P67910 , String:511145.b3619 , UniProt:P67910

Relationship Links: InterPro:IN-FAMILY:IPR001509 , InterPro:IN-FAMILY:IPR011912 , InterPro:IN-FAMILY:IPR016040 , Panther:IN-FAMILY:PTHR10366:SF29 , PDB:Structure:1EQ2 , PDB:Structure:2X6T , Pfam:IN-FAMILY:PF01370

In Paralogous Gene Group: 191 (7 members)

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0009244 - lipopolysaccharide core region biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, Coleman79]
GO:0005975 - carbohydrate metabolic process Inferred by computational analysis [UniProtGOA11a, GOA06, GOA01a]
GO:0006950 - response to stress Inferred by computational analysis [UniProtGOA11a]
GO:0009103 - lipopolysaccharide biosynthetic process Inferred by computational analysis [UniProtGOA11a]
GO:0097171 - ADP-L-glycero-beta-D-manno-heptose biosynthetic process Inferred by computational analysis [UniProtGOA12]
Molecular Function: GO:0005515 - protein binding Inferred from experiment [Butland05]
GO:0008712 - ADP-glyceromanno-heptose 6-epimerase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, Ding94]
GO:0070401 - NADP+ binding Inferred from experiment [Ni01]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01a]
GO:0016853 - isomerase activity Inferred by computational analysis [UniProtGOA11a]
GO:0050661 - NADP binding Inferred by computational analysis [GOA01a]
GO:0050662 - coenzyme binding Inferred by computational analysis [GOA01a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05, Lasserre06]
GO:0016020 - membrane Inferred from experiment [Lasserre06]

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

Essentiality data for rfaD 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 Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Credits:
Last-Curated ? 21-Sep-2010 by Keseler I , SRI International


Enzymatic reaction of: ADP-L-glycero-D-mannoheptose-6-epimerase

Synonyms: AGME

EC Number: 5.1.3.20

ADP-D-glycero-β-D-manno-heptose <=> ADP-L-glycero-β-D-manno-heptose

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is favored in the direction shown.

In Pathways: ADP-L-glycero-β-D-manno-heptose biosynthesis

Summary:
Studies of the catalytic mechanism support a direct C-6'' oxidation/reduction mechanism [Morrison05].

Cofactors or Prosthetic Groups: NADP+ [Ni01]

Inhibitors (Unknown Mechanism): ADP-α-D-glucose [Ding94] , ADP [Ding94]

Kinetic Parameters:

Substrate
Km (μM)
Citations
ADP-D-glycero-β-D-manno-heptose
100.0
[Morrison07, BRENDA14]
ADP-D-glycero-β-D-manno-heptose
100.0
[Ding94, BRENDA14]

T(opt): 42 °C [BRENDA14, Ding94]

pH(opt): 5.5 [BRENDA14, Ding94]


Sequence Features

Feature Class Location Citations Comment
Nucleotide-Phosphate-Binding-Region 10 -> 11
[UniProt10a]
UniProt: NADP;
Nucleotide-Phosphate-Binding-Region 31 -> 32
[UniProt10a]
UniProt: NADP;
Amino-Acid-Sites-That-Bind 38
[UniProt10a]
UniProt: NADP;
Amino-Acid-Sites-That-Bind 53
[UniProt10a]
UniProt: NADP;
Nucleotide-Phosphate-Binding-Region 75 -> 79
[UniProt10a]
UniProt: NADP;
Amino-Acid-Sites-That-Bind 92
[UniProt10a]
UniProt: NADP;
Mutagenesis-Variant 140
[Morrison07, UniProt11]
Alternate sequence: Y → F; UniProt: Severely compromises epimerase activitie.
Active-Site 140
[Morrison07, UniProt08, Morrison07]
 
Amino-Acid-Sites-That-Bind 144
[UniProt10a]
UniProt: NADP;
Amino-Acid-Sites-That-Bind 169
[UniProt10a]
UniProt: Substrate;
Amino-Acid-Sites-That-Bind 170
[UniProt10a]
UniProt: NADP; via amide nitrogen;
Mutagenesis-Variant 178
[Morrison07, UniProt11]
Alternate sequence: K → M; UniProt: Severely compromises epimerase activitie.
Amino-Acid-Sites-That-Bind 178
[UniProt10a]
UniProt: NADP;
Active-Site 178
[Morrison07, UniProt09a, Morrison07]
UniProt: Proton acceptor;
Amino-Acid-Sites-That-Bind 180
[UniProt10a]
UniProt: Substrate; via carbonyl oxygen;
Amino-Acid-Sites-That-Bind 187
[UniProt10a]
UniProt: Substrate;
Protein-Segment 201 -> 204
[UniProt10]
UniProt: Substrate binding; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 208
[Morrison07, UniProt11]
Alternate sequence: K → M; UniProt: Activity similar to that of the wild-type.
Amino-Acid-Sites-That-Bind 209
[UniProt10a]
UniProt: Substrate;
Mutagenesis-Variant 210
[Morrison07, UniProt11]
Alternate sequence: D → N; UniProt: Activity similar to that of the wild-type.
Acetylation-Modification 267
[Zhang09a, UniProt11]
UniProt: N6-acetyllysine.
Amino-Acid-Sites-That-Bind 272
[UniProt10a]
UniProt: Substrate;


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
10/20/97 Gene b3619 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10838; 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

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

Butland05: Butland G, Peregrin-Alvarez JM, Li J, Yang W, Yang X, Canadien V, Starostine A, Richards D, Beattie B, Krogan N, Davey M, Parkinson J, Greenblatt J, Emili A (2005). "Interaction network containing conserved and essential protein complexes in Escherichia coli." Nature 433(7025);531-7. PMID: 15690043

Coleman79: Coleman WG, Leive L (1979). "Two mutations which affect the barrier function of the Escherichia coli K-12 outer membrane." J Bacteriol 139(3);899-910. PMID: 383699

Coleman83: Coleman WG (1983). "The rfaD gene codes for ADP-L-glycero-D-mannoheptose-6-epimerase. An enzyme required for lipopolysaccharide core biosynthesis." J Biol Chem 258(3);1985-90. PMID: 6337148

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

Deacon00: Deacon AM, Ni YS, Coleman WG, Ealick SE (2000). "The crystal structure of ADP-L-glycero-D-mannoheptose 6-epimerase: catalysis with a twist." Structure Fold Des 8(5);453-62. PMID: 10896473

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

Ding94: Ding L, Seto BL, Ahmed SA, Coleman WG (1994). "Purification and properties of the Escherichia coli K-12 NAD-dependent nucleotide diphosphosugar epimerase, ADP-L-glycero-D-mannoheptose 6-epimerase." J Biol Chem 269(39);24384-90. PMID: 7929099

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

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, 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."

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

Joloba04: Joloba ML, Clemmer KM, Sledjeski DD, Rather PN (2004). "Activation of the gab operon in an RpoS-dependent manner by mutations that truncate the inner core of lipopolysaccharide in Escherichia coli." J Bacteriol 186(24);8542-6. PMID: 15576807

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

Karow91: Karow M, Raina S, Georgopoulos C, Fayet O (1991). "Complex phenotypes of null mutations in the htr genes, whose products are essential for Escherichia coli growth at elevated temperatures." Res Microbiol 142(2-3);289-94. PMID: 1656498

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

Kneidinger02: Kneidinger B, Marolda C, Graninger M, Zamyatina A, McArthur F, Kosma P, Valvano MA, Messner P (2002). "Biosynthesis pathway of ADP-L-glycero-beta-D-manno-heptose in Escherichia coli." J Bacteriol 184(2);363-9. PMID: 11751812

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

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

Mayer07: Mayer A, Tanner ME (2007). "Intermediate release by ADP-L-glycero-D-manno-heptose 6-epimerase." Biochemistry 46(20);6149-55. PMID: 17455913

Morrison05: Morrison JP, Read JA, Coleman WG, Tanner ME (2005). "Dismutase activity of ADP-L-glycero-D-manno-heptose 6-epimerase: evidence for a direct oxidation/reduction mechanism." Biochemistry 44(15);5907-15. PMID: 15823050

Morrison07: Morrison JP, Tanner ME (2007). "A two-base mechanism for Escherichia coli ADP-L-glycero-D-manno-heptose 6-epimerase." Biochemistry 46(12);3916-24. PMID: 17316025

Ni01: Ni Y, McPhie P, Deacon A, Ealick S, Coleman WG (2001). "Evidence that NADP+ is the physiological cofactor of ADP-L-glycero-D-mannoheptose 6-epimerase." J Biol Chem 276(29);27329-34. PMID: 11313358

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

Read04: Read JA, Ahmed RA, Morrison JP, Coleman WG, Tanner ME (2004). "The mechanism of the reaction catalyzed by ADP-beta-L-glycero-D-manno-heptose 6-epimerase." J Am Chem Soc 126(29);8878-9. PMID: 15264802

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

UniProt08: UniProt Consortium (2008). "UniProt version 14.6 released on 2008-12-16." Database.

UniProt09a: UniProt Consortium (2009). "UniProt version 15.6 released on 28-JUL-09." Database.

UniProt10: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

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."

Zhang09a: Zhang J, Sprung R, Pei J, Tan X, Kim S, Zhu H, Liu CF, Grishin NV, Zhao Y (2009). "Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli." Mol Cell Proteomics 8(2);215-25. PMID: 18723842

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


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 Sat Nov 22, 2014, biocyc14.