Cloned (Comment) | Organism |
---|---|
gene USP, recombinant expression of His6-biotin-tagged enzyme in Escherichia coli strain BL21(DE3) | Leishmania major |
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 | 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 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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Leishmania major |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Leishmania major | Q4QE75 | - |
- |
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 | 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 | 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 | Comment | Organism |
---|---|---|
UDP-sugar pyrophosphorylase | - |
Leishmania major |
UDP-sugar-producing pyrophosphorylase | - |
Leishmania major |
USP | - |
Leishmania major |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Leishmania major |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Leishmania major |
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 | 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 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 |