Literature summary for 2.3.1.157 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.

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Activating Compound

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

Application

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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
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

Crystallization (Comment)

Organism

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

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

Protein Variants

Protein Variants	Comment	Organism
A451R	site-directed mutagenesis, neither the single mutants A451R and R439T nor the double mutant A451R/R439T affect the acetyltransferase activity significantly	Mycobacterium tuberculosis
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
H374A	site-directed mutagenesis, the mutant shows highly reduced Vmax in acetyltransfer compared to the wild-type enzyme	Mycobacterium tuberculosis
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
K464A	site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme	Mycobacterium tuberculosis
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
K464A/W460A	site-directed mutagenesis, the mutant shows highly compromised activity compared to the wild-type enzyme	Mycobacterium tuberculosis
N397A	site-directed mutagenesis, the mutant shows highly reduced Vmax in acetyltransfer compared to the wild-type enzyme	Mycobacterium tuberculosis
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
R439T	site-directed mutagenesis, neither the single mutants A451R and R439T nor the double mutant A451R/R439T affect the acetyltransferase activity significantly	Mycobacterium tuberculosis
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
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
T418A	site-directed mutagenesis, T418 is the most abundant phosphorylation site on GlmUMtb, acetyltransferase activity is completely abolishe	Mycobacterium tuberculosis
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
T418E	site-directed mutagenesis, acetyltransferase activity of T418E mutant that mimics a phosphorylated Thr, is severely compromised compared to wild-type GlmUMtb	Mycobacterium tuberculosis
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
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
W460A	site-directed mutagenesis, the mutant shows highly compromised activity compared to the wild-type enzyme	Mycobacterium tuberculosis
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
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]

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

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
acetyl-CoA + alpha-D-glucosamine 1-phosphate	Mycobacterium tuberculosis	-	CoA + N-acetyl-alpha-D-glucosamine 1-phosphate	-	?
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	?	-	?
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	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Mycobacterium tuberculosis	-	-	-
Mycobacterium tuberculosis	P9WMN3	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
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

Reaction	Comment	Organism	Reaction ID
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
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
acetyl-CoA + alpha-D-glucosamine 1-phosphate	-	Mycobacterium tuberculosis	CoA + N-acetyl-alpha-D-glucosamine 1-phosphate	-	?
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	-	?
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	?	-	?
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	?	-	?
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	?	-	?

Subunits

Subunits	Comment	Organism
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
trimer	two-domain architecture of GlmU, one monomer per asymmetric unit, and a trimeric quaternary structure known for GlmU proteins	Mycobacterium tuberculosis

Synonyms

Synonyms	Comment	Organism
GlmU	-	Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.6	-	assay at	Mycobacterium tuberculosis

Cofactor

Cofactor	Comment	Organism	Structure
acetyl-CoA	-	Mycobacterium tuberculosis

General Information

General Information	Comment	Organism
evolution	N-acetyl-glucosamine-1-phosphate uridyltransferase, GlmU, is exclusive to prokaryotes, conserved both in Gram positive and Gram negative bacteria	Mycobacterium tuberculosis
malfunction	Deleting the C-terminal tail, i.e. residues 457-495, of GlmUMtb that provides these residues abolishes all acetyltransferase activity	Mycobacterium tuberculosis
metabolism	N-acetyl-glucosamine-1-phosphate uridyltransferase, GlmU, is a bifunctional enzyme involved in bacterial cell wall synthesis	Mycobacterium tuberculosis
additional information	analysis of structures of GlmUMtb bound to substrates of the acetyl transfer reaction	Mycobacterium tuberculosis
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
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
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