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MetaCyc Enzyme: fused N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase

Gene: glmU Accession Numbers: EG11198 (MetaCyc), b3730, ECK3723

Synonyms: yieA

Species: Escherichia coli K-12 substr. MG1655

Subunit composition of fused N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase = [GlmU]3

Summary:
glmU encodes a fused enzyme with two enzymatic activities that catalyze sequential steps in the biosynthesis of UDP-N-acetyl-D-glucosamine (UDP-GlcNAc), an essential precursor of cell wall peptidoglycan, lipopolysaccharide and enterobacterial common antigen [MenginLecreulx94].

The enzymatic activities of GlmU are present in two independently folding and functional domains [Pompeo01]. The uridylyltransferase activity, which catalyzes the formation of UDP-GlcNAc, localizes to the N-terminal domain, and the acetyltransferase activity, which catalyzes the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1-P), is located in the C-terminal domain. Kinetic data shows that the acetyl transfer precedes the uridylyl transfer. The intermediate of the two sequential reactions, N-acetylglucosamine-1-phosphate, appears to be released from the enzyme [Gehring96]. The trimeric organization of the enzyme is required for activity [Pompeo01]. In solution, the enzyme is in a trimer/hexamer equilibrium. Two trimers can interact by a loop in their N-terminal domains that also undergoes a large conformational change during the uridyltransferase reaction [Trempe11].

Inhibitors of GlmU activity, such as iodoacetamide and N-ethyl maleimide, inhibit biofilm formation in urophathogenic E. coli P18 [Burton06]. Inhibitors that are specific for the acetyltransferase activity of GlmU have been identified [Pereira09].

Crystal structures of a truncated form of the enzyme have been solved and show that the protein folds into two distinct domains connected by an α-helical arm [Brown99]. Crystal structures of full-length GlmU together with substrates have been solved, revealing the substrate binding sites and suggesting reaction mechanisms [Olsen01a, Olsen07]. A C307A mutation results in a 1,350-fold decrease in acetyltransferase activity of GlmU [Pompeo98]. A conditional glmU mutant at the restrictive temperature shows morphological changes and cell lysis [MenginLecreulx93]. Both activities of GlmU are required for viability [Brown99, Pompeo01]. A 1-bp insertion into BoxG1 upstream of glmUS was identified as a beneficial adaptation in one population of a long-term evolution experiment [Stanek09].

GlmU: "glucosamine uridyltransferase" [MenginLecreulx93]

Review: [Barreteau08]

Locations: cytosol

Map Position: [3,911,853 <- 3,913,223]

Molecular Weight of Polypeptide: 49.19 kD (from nucleotide sequence), 50.0 kD (experimental) [MenginLecreulx93 ]

Unification Links: ASAP:ABE-0012201 , CGSC:29106 , DIP:DIP-31844N , EchoBASE:EB1184 , EcoGene:EG11198 , EcoliWiki:b3730 , Mint:MINT-1252604 , ModBase:P0ACC7 , OU-Microarray:b3730 , PortEco:glmU , PR:PRO_000022782 , Pride:P0ACC7 , Protein Model Portal:P0ACC7 , RefSeq:NP_418186 , RegulonDB:EG11198 , SMR:P0ACC7 , String:511145.b3730 , UniProt:P0ACC7

Relationship Links: InterPro:IN-FAMILY:IPR001451 , InterPro:IN-FAMILY:IPR005882 , InterPro:IN-FAMILY:IPR011004 , InterPro:IN-FAMILY:IPR018357 , InterPro:IN-FAMILY:IPR025877 , Panther:IN-FAMILY:PTHR22572:SF17 , PDB:Structure:1FWY , PDB:Structure:1FXJ , PDB:Structure:1HV9 , PDB:Structure:2OI5 , PDB:Structure:2OI6 , PDB:Structure:2OI7 , PDB:Structure:3TWD , PDB:Structure:4AA7 , Pfam:IN-FAMILY:PF00132 , Pfam:IN-FAMILY:PF12804 , Prosite:IN-FAMILY:PS00101

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006048 - UDP-N-acetylglucosamine biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, MenginLecreulx93]
GO:0000902 - cell morphogenesis Inferred by computational analysis [GOA06, GOA01a]
GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11a]
GO:0008360 - regulation of cell shape Inferred by computational analysis [UniProtGOA11a]
GO:0009103 - lipopolysaccharide biosynthetic process Inferred by computational analysis [GOA01a]
GO:0009245 - lipid A biosynthetic process Inferred by computational analysis [UniProtGOA12]
GO:0009252 - peptidoglycan biosynthetic process Inferred by computational analysis [UniProtGOA11a, GOA06, GOA01a]
GO:0071555 - cell wall organization Inferred by computational analysis [UniProtGOA11a]
Molecular Function: GO:0000287 - magnesium ion binding Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, MenginLecreulx94]
GO:0003977 - UDP-N-acetylglucosamine diphosphorylase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, Gehring96]
GO:0019134 - glucosamine-1-phosphate N-acetyltransferase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, MenginLecreulx94]
GO:0042802 - identical protein binding Inferred from experiment [Trempe11]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
GO:0016746 - transferase activity, transferring acyl groups Inferred by computational analysis [UniProtGOA11a]
GO:0016779 - nucleotidyltransferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA06, GOA01a]

MultiFun Terms: cell structure murein
cell structure surface antigens (ECA, O antigen of LPS)
metabolism biosynthesis of macromolecules (cellular constituents) enterobacterial common antigen (surface glycolipid)
metabolism biosynthesis of macromolecules (cellular constituents) lipopolysaccharide O antigen
metabolism biosynthesis of macromolecules (cellular constituents) murein (peptidoglycan)
metabolism central intermediary metabolism sugar nucleotide biosynthesis, conversions

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International


Enzymatic reaction of: N-acetylglucosamine-1-phosphate uridyltransferase

Synonyms: UDP-N-acetylglucosamine pyrophosphorylase, UTP:N-acetyl-α-D-glucosamine-1-phosphate uridylyltransferase

EC Number: 2.7.7.23

N-acetyl-α-D-glucosamine 1-phosphate + UTP + H+ <=> UDP-N-acetyl-α-D-glucosamine + diphosphate

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 physiologically favored in the direction shown.

In Pathways: superpathway of UDP-N-acetylglucosamine-derived O-antigen building blocks biosynthesis , O-antigen building blocks biosynthesis (E. coli) , UDP-N-acetyl-D-glucosamine biosynthesis I , anhydromuropeptides recycling

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Summary:
The reaction appears reversible in vitro, with the equilibrium constant estimated to be between 0.15 and 0.3. However, in vivo, the reaction is expected to be unidirectional due to the removal of diphosphate [MenginLecreulx94].

The substrate specificity of a truncated protein containing only the N-terminal uridyltransferase domain has been determined. Results with the full-length protein are similar [Fang09].

Citations: [Guan09]

Cofactors or Prosthetic Groups: Mg2+ [Comment 1, MenginLecreulx94]

Kinetic Parameters:

Substrate
Km (μM)
Citations
N-acetyl-α-D-glucosamine 1-phosphate
18.0
[Gehring96]
UTP
17.0
[Gehring96]

pH(opt): 8.2 [MenginLecreulx94]


Enzymatic reaction of: glucosamine-1-phosphate acetyltransferase

EC Number: 2.3.1.157

D-glucosamine 1-phosphate + acetyl-CoA <=> N-acetyl-α-D-glucosamine 1-phosphate + coenzyme A + H+

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 physiologically favored in the direction shown.

Alternative Substrates for D-glucosamine 1-phosphate [Comment 2 ]: galactosamine-1-phosphate [Gehring96 ] , UDP-α-D-glucosamine [Gehring96 ]

Alternative Substrates for acetyl-CoA [Comment 2 ]: propanoyl-CoA [Gehring96 ]

In Pathways: superpathway of UDP-N-acetylglucosamine-derived O-antigen building blocks biosynthesis , O-antigen building blocks biosynthesis (E. coli) , UDP-N-acetyl-D-glucosamine biosynthesis I , anhydromuropeptides recycling

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Summary:
There is conflicting evidence regarding inhibitors of the enzyme. [MenginLecreulx94] reported that UDP-N-acetylmuramate is the best inhibitor tested, leading to 89% inhibition at 0.1 mM, whereas [Gehring96] found no inhibition by either 0.1 mM UDP-N-acetylmuramate or 10 mM N-acetylmuramate.

The product of the reaction, GlcNAc-1-P, shows 50% inhibition at 0.1 mM [MenginLecreulx94].

The Km for the alternative substrates galactosamine-1-phosphate is 15 mM and for propionyl-CoA 840 µM [Gehring96].

Cofactors or Prosthetic Groups: Mg2+ [Comment 3, MenginLecreulx94]

Inhibitors (Competitive): N-acetyl-α-D-glucosamine 1-phosphate [MenginLecreulx94]

Inhibitors (Unknown Mechanism): iodoacetamide [Pompeo98] , p-hydroxymercuribenzoate [Pompeo98] , N-ethylmaleimide [Pompeo98] , 2-nitro-5-thiocyanobenzoate [Pompeo98, Comment 4] , 5,5'-dithio-bis-2-nitrobenzoate [Pompeo98, Comment 4] , N-acetylmuramate [MenginLecreulx94, Comment 5] , dihydro-UDP-N-acetylmuramate [MenginLecreulx94, Comment 5] , N-acetylmuramic acid 1-phosphate [MenginLecreulx94, Comment 5] , UDP-N-α-D-acetylmuramate [MenginLecreulx94, Comment 6]

Primary Physiological Regulators of Enzyme Activity: N-acetyl-α-D-glucosamine 1-phosphate

Kinetic Parameters:

Substrate
Km (μM)
Citations
acetyl-CoA
600.0
[MenginLecreulx94]
D-glucosamine 1-phosphate
150.0
[MenginLecreulx94]

pH(opt): 8.2 [MenginLecreulx94]


Sequence Features

Feature Class Location Citations Comment
Protein-Segment 1 -> 229
[UniProt10a]
UniProt: Pyrophosphorylase; Sequence Annotation Type: region of interest;
Protein-Segment 11 -> 14
[UniProt10a]
UniProt: Substrate binding; Sequence Annotation Type: region of interest; Non-Experimental Qualifier: by similarity;
Mutagenesis-Variant 14
[Brown99, UniProt11]
Alternate sequence: A; UniProt: 8-fold decrease in uridyltransferase activity. Creates steric conflict and decreases affinity for UTP.
Mutagenesis-Variant 18
[Brown99, UniProt11]
Alternate sequence: A; UniProt: Dramatically impairs the uridyltransferase activity.
Amino-Acid-Sites-That-Bind 25
[UniProt13]
UniProt: UDP-GlcNAc; Non-Experimental Qualifier: by similarity.
Mutagenesis-Variant 25
[Brown99, UniProt11]
Alternate sequence: A; UniProt: 8-fold decrease in uridyltransferase activity.
Amino-Acid-Sites-That-Bind 76
[UniProt10]
UniProt: Substrate;
Protein-Segment 81 -> 82
[UniProt10a]
UniProt: Substrate binding; Sequence Annotation Type: region of interest; Non-Experimental Qualifier: by similarity;
Protein-Segment 103 -> 105
[UniProt13]
UniProt: UDP-GlcNAc binding; Sequence Annotation Type: region of interest.
Metal-Binding-Site 105
[UniProt10]
UniProt: Magnesium;
Amino-Acid-Sites-That-Bind 140
[UniProt13]
UniProt: UDP-GlcNAc; via amide nitrogen.
Amino-Acid-Sites-That-Bind 154
[UniProt10]
UniProt: Substrate;
Amino-Acid-Sites-That-Bind 169
[UniProt10]
UniProt: Substrate;
Sequence-Conflict 186 -> 187
[Walker84, UniProt10]
Alternate sequence: NV; UniProt: (in Ref. 1; CAA25784);
Metal-Binding-Site 227
[UniProt10]
UniProt: Magnesium;
Protein-Segment 230 -> 250
[UniProt10a]
UniProt: Linker; Sequence Annotation Type: region of interest;
Protein-Segment 251 -> 456
[UniProt10a]
UniProt: N-acetyltransferase; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 296
[Pompeo98, UniProt11]
Alternate sequence: A; UniProt: No effect.
Mutagenesis-Variant 307
[Pompeo98, UniProt11]
Alternate sequence: A; UniProt: 1350-fold decrease in acetyltransferase activity.
Active-Site 307
[Pompeo98]
 
Mutagenesis-Variant 324
[Pompeo98, UniProt11]
Alternate sequence: A; UniProt: 8-fold decrease in acetyltransferase activity.
Amino-Acid-Sites-That-Bind 333
[UniProt13]
UniProt: UDP-GlcNAc.
Amino-Acid-Sites-That-Bind 351
[UniProt13]
UniProt: UDP-GlcNAc.
Active-Site 363
[UniProt10a]
UniProt: Proton acceptor; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 366
[UniProt13]
UniProt: UDP-GlcNAc.
Amino-Acid-Sites-That-Bind 377
[UniProt13]
UniProt: UDP-GlcNAc.
Amino-Acid-Sites-That-Bind 380
[UniProt13]
UniProt: Acetyl-CoA; via amide nitrogen.
Mutagenesis-Variant 385
[Pompeo98, UniProt11]
Alternate sequence: A; UniProt: No effect.
Protein-Segment 386 -> 387
[UniProt13]
UniProt: Acetyl-CoA binding; Sequence Annotation Type: region of interest.
Amino-Acid-Sites-That-Bind 405
[UniProt10]
UniProt: Acetyl-CoA;
Amino-Acid-Sites-That-Bind 423
[UniProt10]
UniProt: Acetyl-CoA; via amide nitrogen;
Amino-Acid-Sites-That-Bind 440
[UniProt10]
UniProt: Acetyl-CoA;

History:
10/20/97 Gene b3730 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11198; confirmed by SwissProt match.


References

Barreteau08: Barreteau H, Kovac A, Boniface A, Sova M, Gobec S, Blanot D (2008). "Cytoplasmic steps of peptidoglycan biosynthesis." FEMS Microbiol Rev 32(2);168-207. PMID: 18266853

Brown99: Brown K, Pompeo F, Dixon S, Mengin-Lecreulx D, Cambillau C, Bourne Y (1999). "Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily." EMBO J 18(15);4096-107. PMID: 10428949

Burton06: Burton E, Gawande PV, Yakandawala N, LoVetri K, Zhanel GG, Romeo T, Friesen AD, Madhyastha S (2006). "Antibiofilm activity of GlmU enzyme inhibitors against catheter-associated uropathogens." Antimicrob Agents Chemother 50(5);1835-40. PMID: 16641457

De96a: De Luca C, Lansing M, Crescenzi F, Martini I, Shen GJ, O'Regan M, Wong CH (1996). "Overexpression, one-step purification and characterization of UDP-glucose dehydrogenase and UDP-N-acetylglucosamine pyrophosphorylase." Bioorg Med Chem 4(1);131-41. PMID: 8689233

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

Fang09: Fang J, Guan W, Cai L, Gu G, Liu X, Wang PG (2009). "Systematic study on the broad nucleotide triphosphate specificity of the pyrophosphorylase domain of the N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12." Bioorg Med Chem Lett 19(22);6429-32. PMID: 19804974

Gehring96: Gehring AM, Lees WJ, Mindiola DJ, Walsh CT, Brown ED (1996). "Acetyltransfer precedes uridylyltransfer in the formation of UDP-N-acetylglucosamine in separable active sites of the bifunctional GlmU protein of Escherichia coli." Biochemistry 1996;35(2);579-85. PMID: 8555230

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."

Guan09: Guan W, Cai L, Fang J, Wu B, George Wang P (2009). "Enzymatic synthesis of UDP-GlcNAc/UDP-GalNAc analogs using N-acetylglucosamine 1-phosphate uridyltransferase (GlmU)." Chem Commun (Camb) (45);6976-8. PMID: 19904366

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

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

MenginLecreulx93: Mengin-Lecreulx D, van Heijenoort J (1993). "Identification of the glmU gene encoding N-acetylglucosamine-1-phosphate uridyltransferase in Escherichia coli." J Bacteriol 1993;175(19);6150-7. PMID: 8407787

MenginLecreulx94: Mengin-Lecreulx D, van Heijenoort J (1994). "Copurification of glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities of Escherichia coli: characterization of the glmU gene product as a bifunctional enzyme catalyzing two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis." J Bacteriol 1994;176(18);5788-95. PMID: 8083170

Olsen01a: Olsen LR, Roderick SL (2001). "Structure of the Escherichia coli GlmU pyrophosphorylase and acetyltransferase active sites." Biochemistry 40(7);1913-21. PMID: 11329257

Olsen07: Olsen LR, Vetting MW, Roderick SL (2007). "Structure of the E. coli bifunctional GlmU acetyltransferase active site with substrates and products." Protein Sci 16(6);1230-5. PMID: 17473010

Pereira09: Pereira MP, Blanchard JE, Murphy C, Roderick SL, Brown ED (2009). "High-throughput screening identifies novel inhibitors of the acetyltransferase activity of Escherichia coli GlmU." Antimicrob Agents Chemother 53(6);2306-11. PMID: 19349513

Pompeo01: Pompeo F, Bourne Y, van Heijenoort J, Fassy F, Mengin-Lecreulx D (2001). "Dissection of the bifunctional Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase enzyme into autonomously functional domains and evidence that trimerization is absolutely required for glucosamine-1-phosphate acetyltransferase activity and cell growth." J Biol Chem 276(6);3833-9. PMID: 11084021

Pompeo98: Pompeo F, van Heijenoort J, Mengin-Lecreulx D (1998). "Probing the role of cysteine residues in glucosamine-1-phosphate acetyltransferase activity of the bifunctional GlmU protein from Escherichia coli: site-directed mutagenesis and characterization of the mutant enzymes." J Bacteriol 1998;180(18);4799-803. PMID: 9733680

Stanek09: Stanek MT, Cooper TF, Lenski RE (2009). "Identification and dynamics of a beneficial mutation in a long-term evolution experiment with Escherichia coli." BMC Evol Biol 9;302. PMID: 20040094

Trempe11: Trempe JF, Shenker S, Kozlov G, Gehring K (2011). "Self-association studies of the bifunctional N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli." Protein Sci 20(4);745-52. PMID: 21370307

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

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

UniProt13: UniProt Consortium (2013). "UniProt version 2013-08 released on 2013-08-01 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

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."

Walker84: Walker JE, Gay NJ, Saraste M, Eberle AN (1984). "DNA sequence around the Escherichia coli unc operon. Completion of the sequence of a 17 kilobase segment containing asnA, oriC, unc, glmS and phoS." Biochem J 224(3);799-815. PMID: 6395859


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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
Page generated by SRI International Pathway Tools version 18.5 on Fri Nov 28, 2014, BIOCYC13B.