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Literature summary extracted from

  • Jagtap, P.K.; Soni, V.; Vithani, N.; Jhingan, G.D.; Bais, V.S.; Nandicoori, V.K.; Prakash, B.
    Substrate bound crystal structures reveal features unique to Mycobacterium tuberculosis N-acetyl-glucosamine-1-phosphate uridyltransferase and a catalytic mechanism for acetyltransfer (2012), J. Biol. Chem., 287, 39524-39537.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
2.3.1.157 additional information the enzyme is regulated by PknB via phosphorylation at Thr418 causing downregulation of acetyltransferase Mycobacterium tuberculosis

Application

EC Number Application Comment Organism
2.3.1.157 drug development the enzyme is a target for inhibitor development in treatment of tuberculosis Mycobacterium tuberculosis
2.3.1.157 drug development the enzyme GlmU is a target for development of antibacterial drugs Mycobacterium tuberculosis
2.7.7.23 drug development the enzyme GlmU is a target for development of antibacterial drugs Mycobacterium tuberculosis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.3.1.157 GlmUMtb in complex with substrates/products bound at the acetyltransferase active site, sitting drop vapor diffusion method, mixing of 400 nl of 15 mg/ml protein, 5 mM acetyl-CoA, 5 mM MgCl2, 5 mM UDP-GlcNAc with 400 nl of 18% PEG 3350, 0.1 M Tris-Cl, pH 8.5, and 2% tacsimate, 4-8 days, for enzyme complex with CoA and N-acetylglucosamine-1-phosphate, acetyl-Coa-containing crystals are soaked in 5 mM GlcN-1-P, 5 mM MgCl2, 5 mM UDP-GlcNAc, 5 mM acetyl-CoA, 18% PEG 3350, 0.1 M Tris-Cl, pH 8.5, and 2% tacsimate, or by co-crystallizing the enzyme with 5 mM GlcNAc-1-P, 5 mM MgCl2, 5 mM UDPGlcNAc, and 5 mM CoA under the conditions mentioned for obtaining GlmUMtb(AcCoA) crystals, X-ray diffraction structure determination and analysis at 1.98-2.33 A resolution Mycobacterium tuberculosis
2.3.1.157 purified GlmU, sitting drop vapor diffusion method, mixing of 400 nl of 15 mg/ml GlmU in 5 mM acetyl-CoA, 5 mM MgCl2, 5 mM UDP-GlcNAc with 400 nL of 18% PEG 3350, 0.1 M Tris-Cl, pH 8.5, and 2% tacsimate, 7-8 days, for coupling to acetyl-CoA, crystals are soaked in 5 mM GlcN-1-P, 5 mM MgCl2, 5 mM UDP-GlcNAc, 5 mM acetyl-CoA, 18% PEG 3350, 0.1 M Tris-Cl, pH 8.5, and 2% tacsimate, or in 5 mM GlcNAc-1-P, 5 mM MgCl2, 5 mM UDP-GlcNAc, 5 mM CoA, X-ray diffraction structure determination and analysis at 1.98-2.33 A resolution Mycobacterium tuberculosis
2.7.7.23 GlmUMtb in complex with substrates/products bound at the acetyltransferase active site, sitting drop vapor diffusion method, mixing of 400 nl of 15 mg/ml protein, 5 mM acetyl-CoA, 5 mM MgCl2, 5 mM UDP-GlcNAc with 400 nl of 18% PEG 3350, 0.1 M Tris-Cl, pH 8.5, and 2% tacsimate, 4-8 days, for enzyme complex with CoA and N-acetylglucosamine-1-phosphate, acetyl-Coa-containing crystals are soaked in 5 mM GlcN-1-P, 5 mM MgCl2, 5 mM UDP-GlcNAc, 5 mM acetyl-CoA, 18% PEG 3350, 0.1 M Tris-Cl, pH 8.5, and 2% tacsimate, or by co-crystallizing the enzyme with 5 mM GlcNAc-1-P, 5 mM MgCl2, 5 mM UDPGlcNAc, and 5 mM CoA under the conditions mentioned for obtaining GlmUMtb(AcCoA) crystals, X-ray diffraction structure determination and analysis at 1.98-2.33 A resolution Mycobacterium tuberculosis

Protein Variants

EC Number Protein Variants Comment Organism
2.3.1.157 A451R site-directed mutagenesis, neither the single mutants A451R and R439T nor the double mutant A451R/R439T affect the acetyltransferase activity significantly Mycobacterium tuberculosis
2.3.1.157 A451R/R439T site-directed mutagenesis, neither the single mutants A451R and R439T nor the double mutant A451R/R439T affect the acetyltransferase activity significantly Mycobacterium tuberculosis
2.3.1.157 H374A site-directed mutagenesis, the mutant shows highly reduced Vmax in acetyltransfer compared to the wild-type enzyme Mycobacterium tuberculosis
2.3.1.157 H374A site-directed mutagenesis, the acetyltransferase active site mutant shows 1.7% of acetyltransferase activity and 96.7% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.3.1.157 K464A site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme Mycobacterium tuberculosis
2.3.1.157 K464A site-directed mutagenesis, the mutant still shows acetyltransferase activity, the mutant shows 105.6% acetyltransferase activity and 97.9% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.3.1.157 K464A/W460A site-directed mutagenesis, the mutant shows highly compromised activity compared to the wild-type enzyme Mycobacterium tuberculosis
2.3.1.157 N397A site-directed mutagenesis, the mutant shows highly reduced Vmax in acetyltransfer compared to the wild-type enzyme Mycobacterium tuberculosis
2.3.1.157 N397A site-directed mutagenesis, the acetyltransferase active site mutant shows 5.2% of acetyltransferase activity and 113.6% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.3.1.157 R439T site-directed mutagenesis, neither the single mutants A451R and R439T nor the double mutant A451R/R439T affect the acetyltransferase activity significantly Mycobacterium tuberculosis
2.3.1.157 S416A site-directed mutagenesis, the mutant shows kinetics in acetyltransfer similar to the wild-type enzyme, S416 neither plays a role in catalysis nor in substrate binding Mycobacterium tuberculosis
2.3.1.157 S416A site-directed mutagenesis, the acetyltransferase active site mutant shows 100.9% of acetyltransferase activity and 96.4% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.3.1.157 T418A site-directed mutagenesis, T418 is the most abundant phosphorylation site on GlmUMtb, acetyltransferase activity is completely abolishe Mycobacterium tuberculosis
2.3.1.157 T418A site-directed mutagenesis, the acetyltransferase activity of mutant is severely compromised as compared with GlmUMtb wild-type, the mutant shows 2.4% acetyltransferase activity and 100.4% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.3.1.157 T418E site-directed mutagenesis, acetyltransferase activity of T418E mutant that mimics a phosphorylated Thr, is severely compromised compared to wild-type GlmUMtb Mycobacterium tuberculosis
2.3.1.157 T418E site-directed mutagenesis, the acetyltransferase activity of the T418E mutant that mimics a phosphorylated Thr, is severely compromised as compared with GlmUMtb wild-type, the mutant shows 2.2% acetyltransferase activity and 109.2% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.3.1.157 T418S site-directed mutagenesis, the acetyltransferase activity of the mutant is compromised as compared with GlmUMtb wild-type, the mutant shows 19% acetyltransferase activity and 108.8% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.3.1.157 W460A site-directed mutagenesis, the mutant shows highly compromised activity compared to the wild-type enzyme Mycobacterium tuberculosis
2.3.1.157 W460A site-directed mutagenesis, the mutant displays almost complete loss in acetyltransferase activity, the mutant shows 8.4% acetyltransferase activity and 99.8% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.3.1.157 W460A/K64A site-directed mutagenesis, the mutant shows 7.8% acetyltransferase activity and 104.7% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.7.7.23 H374A site-directed mutagenesis, the acetyltransferase active site mutant shows 1.7% of acetyltransferase activity and 96.7% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.7.7.23 K464A site-directed mutagenesis, the mutant still shows acetyltransferase activity, the mutant shows 105.6% acetyltransferase activity and 97.9% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.7.7.23 N397A site-directed mutagenesis, the acetyltransferase active site mutant shows 5.2% of acetyltransferase activity and 113.6% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.7.7.23 S416A site-directed mutagenesis, the acetyltransferase active site mutant shows 100.9% of acetyltransferase activity and 96.4% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.7.7.23 T418A site-directed mutagenesis, the acetyltransferase activity of mutant is severely compromised as compared with GlmUMtb wild-type, the mutant shows 2.4% acetyltransferase activity and 100.4% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.7.7.23 T418E site-directed mutagenesis, the acetyltransferase activity of the T418E mutant that mimics a phosphorylated Thr, is severely compromised as compared with GlmUMtb wild-type, the mutant shows 2.2% acetyltransferase activity and 109.2% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.7.7.23 T418S site-directed mutagenesis, the acetyltransferase activity of the mutant is compromised as compared with GlmUMtb wild-type, the mutant shows 19% acetyltransferase activity and 108.8% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.7.7.23 W460A site-directed mutagenesis, the mutant displays almost complete loss in acetyltransferase activity, the mutant shows 8.4% acetyltransferase activity and 99.8% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis
2.7.7.23 W460A/K64A site-directed mutagenesis, the mutant shows 7.8% acetyltransferase activity and 104.7% of uridinyltransferase activity compared to the wild-type Mycobacterium tuberculosis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.3.1.157 0.283
-
 alpha-D-glucosamine 1-phosphate pH 7.6, 30°C, recombinant mutant N397A Mycobacterium tuberculosis
2.3.1.157 0.3
-
 acetyl-CoA pH 7.6, 30°C, recombinant mutant H374A Mycobacterium tuberculosis
2.3.1.157 0.3
-
 alpha-D-glucosamine 1-phosphate pH 7.6, 30°C, recombinant mutant H374A Mycobacterium tuberculosis
2.3.1.157 0.353
-
 alpha-D-glucosamine 1-phosphate pH 7.6, 30°C, recombinant wild-type Mycobacterium tuberculosis
2.3.1.157 0.355
-
 acetyl-CoA pH 7.6, 30°C, recombinant mutant N397A Mycobacterium tuberculosis
2.3.1.157 0.355
-
 acetyl-CoA pH 7.6, 30°C, recombinant mutant S416A Mycobacterium tuberculosis
2.3.1.157 0.355
-
 acetyl-CoA pH 7.6, 30°C, recombinant wild-type Mycobacterium tuberculosis
2.3.1.157 0.37
-
 alpha-D-glucosamine 1-phosphate pH 7.6, 30°C, recombinant mutant S416A Mycobacterium tuberculosis

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.7.23 Mg2+ required Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.3.1.157 acetyl-CoA + alpha-D-glucosamine 1-phosphate Mycobacterium tuberculosis
-
 CoA + N-acetyl-alpha-D-glucosamine 1-phosphate
-
 ?
2.3.1.157 additional information Mycobacterium tuberculosis N-acetyl-glucosamine-1-phosphate uridyltransferase, GlmU, a bifunctional enzyme that catalyzes two key reactions: acetyltransfer and uridyltransfer at two independent domains, overview ?
-
 ?
2.3.1.157 additional information Mycobacterium tuberculosis N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme catalyzing the reactions of EC 2.3.1.157, N-acetylglucosamine-1-phosphate uridyltransferase, and 2.7.7.23, UDP-N-acetylglucosamine diphosphorylase, the enzyme catalyzes the two reactions, acetyl transfer and uridyl transfer, at two independent domains, regulation, overview ?
-
 ?
2.7.7.23 additional information Mycobacterium tuberculosis N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme catalyzing the reactions of EC 2.3.1.157, N-acetylglucosamine-1-phosphate uridyltransferase, and 2.7.7.23, UDP-N-acetylglucosamine diphosphorylase, the enzyme catalyzes the two reactions, acetyl transfer and uridyl transfer, at two independent domains, regulation, overview ?
-
 ?
2.7.7.23 UTP + N-acetyl-alpha-D-glucosamine 1-phosphate Mycobacterium tuberculosis
-
 diphosphate + UDP-N-acetyl-alpha-D-glucosamine
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.3.1.157 Mycobacterium tuberculosis
-
-
-
2.3.1.157 Mycobacterium tuberculosis P9WMN3
-
-
2.7.7.23 Mycobacterium tuberculosis P9WMN3
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
2.3.1.157 phosphoprotein the enzyme is regulated by PknB via phosphorylation at Thr418 causing downregulation of acetyltransferase activity leaving its uridyltransferase activity unaffected, identification of phosphorylation site by mass spectrometry Mycobacterium tuberculosis
2.7.7.23 phosphoprotein the enzyme is regulated by PknB via phosphorylation at Thr418 causing downregulation of acetyltransferase activity leaving its uridyltransferase activity unaffected, identification of phosphorylation site by mass spectrometry Mycobacterium tuberculosis

Reaction

EC Number Reaction Comment Organism Reaction ID
2.3.1.157 acetyl-CoA + alpha-D-glucosamine 1-phosphate = CoA + N-acetyl-alpha-D-glucosamine 1-phosphate substrate recognition, and catalytic mechanism for acetyltransfer involving His374, Asn397 and Ala391, overview Mycobacterium tuberculosis
a
2.3.1.157 acetyl-CoA + alpha-D-glucosamine 1-phosphate = CoA + N-acetyl-alpha-D-glucosamine 1-phosphate the catalytic mechanism is a SN2, bimolecular nucleophilic substitution reaction, catalyzed by the C-terminal domain. His374 and Asn397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser416 and Trp460, on a short helix, provide important interactions for substrate binding Mycobacterium tuberculosis
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.3.1.157 acetyl-CoA + alpha-D-glucosamine 1-phosphate
-
 Mycobacterium tuberculosis CoA + N-acetyl-alpha-D-glucosamine 1-phosphate
-
 ?
2.3.1.157 acetyl-CoA + alpha-D-glucosamine 1-phosphate uncommon mode of acetyl-CoA binding in GlmUMtb in the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Higly conserved Trp460 is critical for acetyl-CoA binding Mycobacterium tuberculosis CoA + N-acetyl-alpha-D-glucosamine 1-phosphate
-
 ?
2.3.1.157 additional information N-acetyl-glucosamine-1-phosphate uridyltransferase, GlmU, a bifunctional enzyme that catalyzes two key reactions: acetyltransfer and uridyltransfer at two independent domains, overview Mycobacterium tuberculosis ?
-
 ?
2.3.1.157 additional information N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme catalyzing the reactions of EC 2.3.1.157, N-acetylglucosamine-1-phosphate uridyltransferase, and 2.7.7.23, UDP-N-acetylglucosamine diphosphorylase, the enzyme catalyzes the two reactions, acetyl transfer and uridyl transfer, at two independent domains, regulation, overview Mycobacterium tuberculosis ?
-
 ?
2.3.1.157 additional information coupled assay method: coupling of the two enzyme reactions via N-acetyl-alpha-D-glucosamine 1-phosphate for determination of the acetyl transferase activity of the enzyme. Substrate recognition and catalytic mechanism for acetyl transfer, overview Mycobacterium tuberculosis ?
-
 ?
2.7.7.23 additional information N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme catalyzing the reactions of EC 2.3.1.157, N-acetylglucosamine-1-phosphate uridyltransferase, and 2.7.7.23, UDP-N-acetylglucosamine diphosphorylase, the enzyme catalyzes the two reactions, acetyl transfer and uridyl transfer, at two independent domains, regulation, overview Mycobacterium tuberculosis ?
-
 ?
2.7.7.23 additional information coupled assay method: coupling of the two enzyme reactions via N-acetyl-alpha-D-glucosamine 1-phosphate for determination of the acetyl transferase activity of the enzyme Mycobacterium tuberculosis ?
-
 ?
2.7.7.23 UTP + N-acetyl-alpha-D-glucosamine 1-phosphate
-
 Mycobacterium tuberculosis diphosphate + UDP-N-acetyl-alpha-D-glucosamine
-
 ?

Subunits

EC Number Subunits Comment Organism
2.3.1.157 trimer GlmU has a conserved two-domain architecture, one monomer per asymmetric unit, and a trimeric quaternary structure, modeling of acetyl-CoA bound to the C-terminal domain, overview Mycobacterium tuberculosis
2.3.1.157 trimer two-domain architecture of GlmU, one monomer per asymmetric unit, and a trimeric quaternary structure known for GlmU proteins Mycobacterium tuberculosis
2.7.7.23 trimer two-domain architecture of GlmU, one monomer per asymmetric unit, and a trimeric quaternary structure known for GlmU proteins Mycobacterium tuberculosis

Synonyms

EC Number Synonyms Comment Organism
2.3.1.157 GlmU
-
 Mycobacterium tuberculosis
2.7.7.23 GlmU
-
 Mycobacterium tuberculosis
2.7.7.23 N-acetyl-glucosamine-1-phosphate uridyltransferase
-
 Mycobacterium tuberculosis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.3.1.157 30
-
 assay at Mycobacterium tuberculosis
2.7.7.23 30
-
 assay at Mycobacterium tuberculosis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.3.1.157 7.6
-
 assay at Mycobacterium tuberculosis
2.7.7.23 7.6
-
 assay at Mycobacterium tuberculosis

Cofactor

EC Number Cofactor Comment Organism Structure
2.3.1.157 acetyl-CoA
-
 Mycobacterium tuberculosis

General Information

EC Number General Information Comment Organism
2.3.1.157 evolution N-acetyl-glucosamine-1-phosphate uridyltransferase, GlmU, is exclusive to prokaryotes, conserved both in Gram positive and Gram negative bacteria Mycobacterium tuberculosis
2.3.1.157 malfunction Deleting the C-terminal tail, i.e. residues 457-495, of GlmUMtb that provides these residues abolishes all acetyltransferase activity Mycobacterium tuberculosis
2.3.1.157 metabolism N-acetyl-glucosamine-1-phosphate uridyltransferase, GlmU, is a bifunctional enzyme involved in bacterial cell wall synthesis Mycobacterium tuberculosis
2.3.1.157 additional information analysis of structures of GlmUMtb bound to substrates of the acetyl transfer reaction Mycobacterium tuberculosis
2.3.1.157 additional information the catalytic mechanism operative in GlmUMtb performs a SN2 reaction, His374 and Asn397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser416 and Trp460, on a short helix, provide important interactions for substrate binding. The enzyme shows an uncommon mode of binding with acetyl-CoA. GlmU from Mycobacterium tuberculosis possesses a unique 30-residue extension at the C-terminus. The adenine base of acetyl-CoA bound to GlmUMtb is buried at the interface of two monomers of the trimer Mycobacterium tuberculosis
2.3.1.157 physiological function GlmU is involved in the biosynthesis of UDP-N-acetylglucosamine-1-phosphate. It is a bifunctional protein with two independent active sites catalyzing acetyl transfer and uridyl transfer reactions on glucosamine-1-phosphate. It synthesizes two key intermediates of cell wall biosynthesis pathways, viz. N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and UDP-GlcNAc. The acetyltransferase activity is catalyzed by the C-terminal domain. It is essential for the growth of the organism Mycobacterium tuberculosis
2.3.1.157 physiological function N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme involved in bacterial cell wall synthesis and is exclusive to prokaryotes. The enzyme is regulated by PknB via phosphorylation at Thr418 causing downregulation of acetyltransferase Mycobacterium tuberculosis
2.7.7.23 additional information GlmU from Mycobacterium tuberculosis possesses a unique 30-residue extension at the C-terminus Mycobacterium tuberculosis
2.7.7.23 physiological function N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) is a bifunctional enzyme involved in bacterial cell wall synthesis and is exclusive to prokaryotes Mycobacterium tuberculosis