Literature summary for 2.7.7.23 extracted from

Urbaniak, M.D.; Collie, I.T.; Fang, W.; Aristotelous, T.; Eskilsson, S.; Raimi, O.G.; Harrison, J.; Navratilova, I.H.; Frearson, J.A.; van Aalten, D.M.; Ferguson, M.A.
A novel allosteric inhibitor of the uridine diphosphate N-acetylglucosamine pyrophosphorylase from Trypanosoma brucei (2013), ACS Chem. Biol., 8, 1981-1987.

Application

Application	Comment	Organism
drug development	the identification of a selective inhibitory allosteric binding site in the parasite enzyme and the fact that the human and trypanosome enzymes both display a strictly ordered bi-bi mechanism, but with the order of substrate binding reversed, makes the enzyme a good target for drug development	Trypanosoma brucei

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Homo sapiens
expression in Escherichia coli	Trypanosoma brucei brucei
recombinant expression of His-tagged enzyme and of GST-tagged in Escherichia coli strain BL21(DE3)	Trypanosoma brucei

Crystallization (Commentary)

Crystallization (Comment)	Organism
in complex with inhibitor 3-[2-(1,3-benzodioxol-5-yl)-2-oxoethyl]-4-bromo-3-hydroxy-5-methyl-1,3-dihydro-2H-indol-2-one. Only the (R)-enantiomer binds, and it is likely that the kinked shape of the molecule is crucial for its shape-complimentarity to the pocket. The benzo[1,3]dioxole moiety is deeply buried, making close contact with Ala397 and Gly232 at the bottom of the cleft. The indolin-2-one sits at the top of the cleft, with the unsubstituted edge exposed to solvent and the methyl and bromide substituents on making contact with Ala239, Met370, Lys371, and Ala367	Trypanosoma brucei brucei
purified recombinant detagged enzyme in complex with inhibitor 1, sitting-drop vapor diffusion method, mixing of 500 nl of 15 mg/ml protein solution wit 500 nl of precipitant solution containing 25% PEG 3350, 0.2 M (NH4)2SO4, 0.1 M Bis-Tris, pH 5.5, 22°C, X-ray diffraction structure determination and analysis at 1.75 A resolution	Trypanosoma brucei

Crystallization (Comment)

Organism

in complex with inhibitor 3-[2-(1,3-benzodioxol-5-yl)-2-oxoethyl]-4-bromo-3-hydroxy-5-methyl-1,3-dihydro-2H-indol-2-one. Only the (R)-enantiomer binds, and it is likely that the kinked shape of the molecule is crucial for its shape-complimentarity to the pocket. The benzo[1,3]dioxole moiety is deeply buried, making close contact with Ala397 and Gly232 at the bottom of the cleft. The indolin-2-one sits at the top of the cleft, with the unsubstituted edge exposed to solvent and the methyl and bromide substituents on making contact with Ala239, Met370, Lys371, and Ala367

Trypanosoma brucei brucei

purified recombinant detagged enzyme in complex with inhibitor 1, sitting-drop vapor diffusion method, mixing of 500 nl of 15 mg/ml protein solution wit 500 nl of precipitant solution containing 25% PEG 3350, 0.2 M (NH4)2SO4, 0.1 M Bis-Tris, pH 5.5, 22°C, X-ray diffraction structure determination and analysis at 1.75 A resolution

Trypanosoma brucei

Inhibitors

Inhibitors	Comment	Organism
3-[2-(1,3-benzodioxol-5-yl)-2-oxoethyl]-4-bromo-3-hydroxy-5-methyl-1,3-dihydro-2H-indol-2-one	competitive inhibitor with selectivity over the human counterpart, binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP	Trypanosoma brucei brucei
3-[2-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-oxoethyl]-3-hydroxy-5-methyl-1,3-dihydro-2H-indol-2-one	-	Trypanosoma brucei
3-[2-(2H-1,3-benzodioxol-5-yl)-2-oxoethyl]-3-hydroxy-6,7-dimethyl-1,3-dihydro-2H-indol-2-one	-	Trypanosoma brucei
3-[2-(2H-1,3-benzodioxol-5-yl)-2-oxoethyl]-3-hydroxy-7-methyl-1,3-dihydro-2H-indol-2-one	-	Trypanosoma brucei
3-[2-(2H-1,3-benzodioxol-5-yl)-2-oxoethyl]-4,6-dichloro-3-hydroxy-1,3-dihydro-2H-indol-2-one	-	Trypanosoma brucei
3-[2H-(1,3-benzodioxol-5-yl)-2-oxoethyl]-4-bromo-3-hydroxy-5-methyl-1,3-dihydro-2H-indol-2-one	UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. The inhibitor binds at an allosteric site	Trypanosoma brucei
additional information	TbUAP inhibitor by high-throughput screening, and structure-activity relationships, overview. No inhibition by 3	Trypanosoma brucei
N-(3,5-dimethoxyphenyl)-2,3-dihydro-4H-1,4-benzothiazine-4-carboxamide	-	Trypanosoma brucei

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	the UAP enzyme kinetics display a strictly ordered bi.bi mechanism	Trypanosoma brucei

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Trypanosoma brucei

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	Q16222	-	-
Trypanosoma brucei	-	-	-
Trypanosoma brucei brucei	Q386Q8	-	-
Trypanosoma brucei brucei 927 / 4 GUTat10.1 / TREU927	Q386Q8	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, recombinant GST-tagged enzyme from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography, tag cleavage, and gel filtration	Trypanosoma brucei

Source Tissue

Source Tissue	Comment	Organism	Textmining
bloodstream form	-	Trypanosoma brucei	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	the human and trypanosome enzymes both display a strictly ordered bi-bi mechanism, but with the order of substrate binding reversed. Human UAP does not bind UTP alone, and although it does show significant binding to GlcNAc-1-P, it is not possible to calculate an affinity due to complex binding kinetics	Homo sapiens	?	-	?
additional information	the human and trypanosome enzymes both display a strictly ordered bi-bi mechanism, but with the order of substrate binding reversed. Trypanosoma brucei UAP binds UTP alone with a KD of 83.1 microM, but does not bind GlcNAc-1-P alone	Trypanosoma brucei brucei	?	-	?
additional information	the human and trypanosome enzymes both display a strictly ordered bi-bi mechanism, but with the order of substrate binding reversed. Trypanosoma brucei UAP binds UTP alone with a KD of 83.1 microM, but does not bind GlcNAc-1-P alone	Trypanosoma brucei brucei 927 / 4 GUTat10.1 / TREU927	?	-	?
UTP + N-acetyl-alpha-D-glucosamine 1-phosphate	-	Trypanosoma brucei	diphosphate + UDP-N-acetyl-alpha-D-glucosamine	-	r

Synonyms

Synonyms	Comment	Organism
Tb11.02.0120	-	Trypanosoma brucei brucei
UAP	-	Trypanosoma brucei
uridine diphosphate N-acetylglucosamine pyrophosphorylase	-	Trypanosoma brucei

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Trypanosoma brucei

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor
0.037	-	pH 7.5, temperature not specified in the publication	Trypanosoma brucei	3-[2H-(1,3-benzodioxol-5-yl)-2-oxoethyl]-4-bromo-3-hydroxy-5-methyl-1,3-dihydro-2H-indol-2-one
0.037	-	pH not specified in the publication, temperature not specified in the publication	Trypanosoma brucei brucei	3-[2-(1,3-benzodioxol-5-yl)-2-oxoethyl]-4-bromo-3-hydroxy-5-methyl-1,3-dihydro-2H-indol-2-one
0.049	-	pH 7.5, temperature not specified in the publication	Trypanosoma brucei	N-(3,5-dimethoxyphenyl)-2,3-dihydro-4H-1,4-benzothiazine-4-carboxamide

General Information

General Information	Comment	Organism
physiological function	the enzyme catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei,	Trypanosoma brucei