Literature summary for 2.7.1.36 extracted from

Sgraja, T.; Smith, T.K.; Hunter, W.N.
Structure, substrate recognition and reactivity of Leishmania major mevalonate kinase (2007), BMC Struct. Biol., 7, 20.

Cloned(Commentary)

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 (Commentary)

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

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

Metals/Ions	Comment	Organism	Structure
Mg2+	-	Leishmania major

Natural Substrates/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Leishmania major	-	-	-
Leishmania major	Q4Q6K7	-	-
Trypanosoma brucei	-	-	-

Purification (Commentary)

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

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	a

Specific Activity [micromol/min/mg]

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 and Products (Substrate)

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 [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay ta	Leishmania major

pH Optimum

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

Cofactor

Cofactor	Comment	Organism
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