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Literature summary for 2.7.7.64 extracted from

  • Prakash, O.; Fuehring, J.; Post, J.; Shepherd, S.M.; Eadsforth, T.C.; Gray, D.; Fedorov, R.; Routier, F.H.
    Identification of Leishmania major UDP-sugar pyrophosphorylase inhibitors using biosensor-based small molecule fragment library screening (2019), Molecules, 24, 996 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene USP, recombinant expression of His6-biotin-tagged enzyme in Escherichia coli strain BL21(DE3) Leishmania major

Protein Variants

Protein Variants Comment Organism
F383R site-directed mutagenesis, the mutant shows 88% reduction of catalytic efficiency compared to the wild-type enzyme enzyme with substrate alpha-D-galactose 1-phosphate Leishmania major
V199W site-directed mutagenesis, the mutant shows 78% reduction of catalytic efficiency compared to the wild-type enzyme with substrate alpha-D-galactose 1-phosphate Leishmania major
V330W site-directed mutagenesis, the mutant shows 38% reduction of catalytic efficiency compared to the wild-type enzyme enzyme with substrate alpha-D-galactose 1-phosphate Leishmania major
V330W/F383R site-directed mutagenesis, the mutant shows 77% reduction of catalytic efficiency compared to the wild-type enzyme enzyme with substrate alpha-D-galactose 1-phosphate Leishmania major

Inhibitors

Inhibitors Comment Organism Structure
(3R)-1-(4-chlorobenzoyl)-N-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)piperidine-3-carboxamide compound (3R)-1-(4-chlorobenzoyl)-N-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)piperidine-3-carboxamide preferentially binds to one site located in the proximity of the active center of the enzyme Leishmania major
2-(5,6-dimethylthieno[2,3-d]pyrimidin-4-yl)oxybenzamide
-
Leishmania major
2-(furan-2-yl)-N'-(indol-3-ylidenemethyl)quinoline-4-carbohydrazide
-
Leishmania major
2-[(3As,7aR)-1,3-dioxo-3a,4,7,7a-tetrahydroisoindol-2-yl]-N-[4-(propan-2-ylsulfamoyl)phenyl]acetamide
-
Leishmania major
2-[3-(trifluoromethyl)anilino]-1,3-thiazol-4-one compound 2-[3-(trifluoromethyl)anilino]-1,3-thiazol-4-on preferentially binds to one site located in the proximity of the active center of the enzyme Leishmania major
3-(2-aminoethyl)-1-methylquinazoline-2,4(1H,3H)-dione
-
Leishmania major
3-(trifluoromethyl)benzoic acid
-
Leishmania major
ethyl 2,4-dimethyl-5-[(2R)-2-[4-(2-phenoxyethyl)piperazin-1-yl]propanoyl]-1H-pyrrole-3-carboxylate
-
Leishmania major
methyl (2R)-2-[[2-(4-fluorophenyl)sulfanylacetyl]amino]-3-hydroxypropanoate
-
Leishmania major
methyl(3-fluorophenyl)acetate
-
Leishmania major
additional information identification of structurally diverse scaffolds for the development of USP inhibitors by fragment library screening, selection of small molecule inhibitors with allosteric regulation sites, that inhibit both Leishmania major USP and UGP. Inhibitor binding kinetics, computational docking study, overview Leishmania major
N-(2-methoxyethyl)-2-oxo-1,3-benzoxazole-3(2H)-carboxamide
-
Leishmania major

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics Leishmania major
0.2765
-
alpha-D-galactose 1-phosphate mutant V330W/F383R, pH and temperature not specified in the publication Leishmania major
0.364
-
alpha-D-galactose 1-phosphate mutant F383Rd-type enzyme, pH and temperature not specified in the publication Leishmania major
0.529
-
alpha-D-galactose 1-phosphate wild-type enzyme, pH and temperature not specified in the publication Leishmania major
1.007
-
alpha-D-galactose 1-phosphate mutant F199W, pH and temperature not specified in the publication Leishmania major
1.255
-
alpha-D-galactose 1-phosphate mutant V330W, pH and temperature not specified in the publication Leishmania major

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Leishmania major

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
UTP + alpha-D-galactose 1-phosphate Leishmania major
-
diphosphate + UDP-alpha-D-galactose
-
r
UTP + alpha-D-glucose 1-phosphate Leishmania major
-
diphosphate + UDP-alpha-D-glucose
-
r

Organism

Organism UniProt Comment Textmining
Leishmania major Q4QE75
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-biotin-tagged enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography, tag cleavage via TEV protease, and dialysis Leishmania major

Reaction

Reaction Comment Organism Reaction ID
UTP + a monosaccharide 1-phosphate = diphosphate + UDP-monosaccharide UDP-sugar-producing pyrophosphorylases catalyze reversible reactions, require magnesium for catalysis, and act through an ordered sequential Bi-Bi reaction mechanism. In the forward reaction, UTP binds before the sugar 1-phosphate, and after catalysis, inorganic diphosphate is released before the UDP-sugar Leishmania major

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
UTP + alpha-D-galactose 1-phosphate
-
Leishmania major diphosphate + UDP-alpha-D-galactose
-
r
UTP + alpha-D-glucose 1-phosphate
-
Leishmania major diphosphate + UDP-alpha-D-glucose
-
r

Synonyms

Synonyms Comment Organism
UDP-sugar pyrophosphorylase
-
Leishmania major
UDP-sugar-producing pyrophosphorylase
-
Leishmania major
USP
-
Leishmania major

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Leishmania major

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
17.33
-
alpha-D-galactose 1-phosphate mutant F383Rd-type enzyme, pH and temperature not specified in the publication Leishmania major
25.58
-
alpha-D-galactose 1-phosphate mutant V330W/F383R, pH and temperature not specified in the publication Leishmania major
90.25
-
alpha-D-galactose 1-phosphate mutant F199W, pH and temperature not specified in the publication Leishmania major
216.45
-
alpha-D-galactose 1-phosphate wild-type enzyme, pH and temperature not specified in the publication Leishmania major
319.32
-
alpha-D-galactose 1-phosphate mutant V330W, pH and temperature not specified in the publication Leishmania major

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Leishmania major

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.0404 0.0463 wild-type enzyme, pH 8.0, 25°C Leishmania major ethyl 2,4-dimethyl-5-[(2R)-2-[4-(2-phenoxyethyl)piperazin-1-yl]propanoyl]-1H-pyrrole-3-carboxylate
0.0472 0.0664 wild-type enzyme, pH 8.0, 25°C Leishmania major methyl (2R)-2-[[2-(4-fluorophenyl)sulfanylacetyl]amino]-3-hydroxypropanoate
0.0781 0.1066 wild-type enzyme, pH 8.0, 25°C Leishmania major (3R)-1-(4-chlorobenzoyl)-N-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)piperidine-3-carboxamide
0.135 0.175 wild-type enzyme, pH 8.0, 25°C Leishmania major 2-[3-(trifluoromethyl)anilino]-1,3-thiazol-4-one
0.1698 0.1948 wild-type enzyme, pH 8.0 25°C Leishmania major 2-(5,6-dimethylthieno[2,3-d]pyrimidin-4-yl)oxybenzamide
0.25
-
above, wild-type enzyme, pH 8.0, 25°C Leishmania major 2-(furan-2-yl)-N'-(indol-3-ylidenemethyl)quinoline-4-carbohydrazide
0.25
-
above, wild-type enzyme, pH 8.0, 25°C Leishmania major 2-[(3As,7aR)-1,3-dioxo-3a,4,7,7a-tetrahydroisoindol-2-yl]-N-[4-(propan-2-ylsulfamoyl)phenyl]acetamide

General Information

General Information Comment Organism
malfunction inactivation of the two parasite diphosphorylases UGP and USP, results in parasite death Leishmania major
metabolism Leishmania parasites possess a UDP-sugar pyrophosphorylase (USP), which has broad substrate specificity and is involved in monosaccharide salvage Leishmania major
physiological function Leishmania parasites express a UDP-sugar pyrophosphorylase (USP) responsible for monosaccharides salvage that is able to generate both UDP-Gal and UDP-Glc. In vitro, Leishmania USP preferentially uses UTP as a nucleotide donor, and can activate a variety of sugar-1-phosphates. The enzyme is most active with galactose 1-phosphate and glucose 1-phosphate, with a slight preference for the former, but it cannot activate N-acetylated sugars. UDP-sugar-producing diphosphorylases catalyze reversible reactions, require magnesium for catalysis, and act through an ordered sequential Bi-Bi reaction mechanism. In the forward reaction, UTP binds before the sugar 1-phosphate, and after catalysis, inorganic diphosphate is released before the UDP-sugar Leishmania major

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
47.61
-
alpha-D-galactose 1-phosphate mutant F383Rd-type enzyme, pH and temperature not specified in the publication Leishmania major
89.62
-
alpha-D-galactose 1-phosphate mutant F199W, pH and temperature not specified in the publication Leishmania major
92.51
-
alpha-D-galactose 1-phosphate mutant V330W/F383R, pH and temperature not specified in the publication Leishmania major
254.44
-
alpha-D-galactose 1-phosphate mutant V330W, pH and temperature not specified in the publication Leishmania major
409.17
-
alpha-D-galactose 1-phosphate wild-type enzyme, pH and temperature not specified in the publication Leishmania major