Cloned (Comment) | Organism |
---|---|
DNA and amino acid sequence determination and anaylsis, overexpression in a procyclic form of Trypanosoma brucei, and expression of His-tagged wild-type enzyme in Escherichia coli strain BL21(DE3), and of His-tagged selenomethionine-labeled enzyme in Escherichia coli strain B834 | Leishmania major |
DNA and amino acid sequence determination and anaylsis, overexprssion in a procyclic form of Trypanosoma brucei | Trypanosoma brucei |
expression in Trypanosoma brucei | Leishmania major |
Crystallization (Comment) | Organism |
---|---|
free enzyme and in complex with mevalonate, 1.75 A and 1.9 A resolution, respectively. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R)- over (S)-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of (R)-mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155 | Leishmania major |
purified recombinant wild-type and selenomethionine-labeled enzyme as apoenzyme and in complex with (R)-mevalonate, hanging drop vapor diffusion method, 0.002 ml of 7.5 mg/ml protein in 10 mM Tris-HCl, pH 8.5, 20 mM NaCl, and 1 mM DTT, in presence of 3-25 mM adenosine 5'-(beta,gamma-imino)triphosphate, 6-50 mM (R)-mevalonate, 10 mM Mg2+, mixing with reservoir solution containing 1.15 M sodium citrate, pH 6.2, 18°C, X-ray diffraction structure determination and analysis at 1.75-1.9 A resolution, single-wavelength anomalous dispersion, molecular replacement fails | Leishmania major |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Leishmania major |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + (R)-mevalonate | Trypanosoma brucei | - |
ADP + (R)-5-phosphomevalonate | - |
r | |
ATP + (R)-mevalonate | Leishmania major | - |
ADP + (R)-5-phosphomevalonate | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Leishmania major | - |
- |
- |
Leishmania major | Q4Q6K7 | - |
- |
Trypanosoma brucei | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, and anion exchange chromatography to homogeneity | Leishmania major |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
ATP + (R)-mevalonate = ADP + (R)-5-phosphomevalonate | catalytic reaction mechanism, substrate binding structure involves key residue His25 in a deep cavity lined by highly conserved residues, Lys18, Asp155, Val202, and Thr283 are involved in catalysis | Leishmania major |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
development of a highly sensitive radioactive assay | Leishmania major |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + (R)-mevalonate | - |
Trypanosoma brucei | ADP + (R)-5-phosphomevalonate | - |
r | |
ATP + (R)-mevalonate | - |
Leishmania major | ADP + (R)-5-phosphomevalonate | - |
r |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay ta | Leishmania major |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Leishmania major |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ADP | - |
Trypanosoma brucei | |
ADP | - |
Leishmania major | |
ATP | - |
Trypanosoma brucei | |
ATP | helix alpha2 and the preceding polypeptide adopt a conformation impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding, the ATP binding structure is distinct from related enzymes, overview | Leishmania major |