Literature summary for 2.7.7.23 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.

Application

Application	Comment	Organism
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

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

Protein Variants

Protein Variants	Comment	Organism
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 still shows acetyltransferase activity, the mutant shows 105.6% acetyltransferase activity and 97.9% of uridinyltransferase activity compared to the wild-type	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
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, 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, 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 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

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
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

Substrates and Products (Substrate)

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

Subunits

Subunits	Comment	Organism
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
N-acetyl-glucosamine-1-phosphate uridyltransferase	-	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

General Information

General Information	Comment	Organism
additional information	GlmU from Mycobacterium tuberculosis possesses a unique 30-residue extension at the C-terminus	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	Mycobacterium tuberculosis