Literature summary for 2.7.1.50 extracted from

Drebes, J.; Kuenz, M.; Windshuegel, B.; Kikhney, A.G.; Mueller, I.B.; Eberle, R.J.; Oberthuer, D.; Cang, H.; Svergun, D.I.; Perbandt, M.; Betzel, C.; Wrenger, C.
Structure of ThiM from vitamin B1 biosynthetic pathway of Staphylococcus aureus - insights into a novel pro-drug approach addressing MRSA infections (2016), Sci. Rep., 6, 22871 .

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme SaThiM in apoform and in complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues, cpd1 and cpd2, sitting drop vapor diffusion method using 10-20 mg/ml protein in 100 mM Tris, and 150 mM NaCl, pH 8.0, with an equal volume of 0.5 M magnesium formate, microcrystal seeding, with precipitant solution containing 18, 20 or 22% PEG 3350 w/v, 0.2 M magnesium formate, and 5% isopropanol (v/v), against a 0.5 ml reservoir of precipitant, X-ray diffraction structure determination and analysis at 1.62-2.09 A resolution. The homologue structure of Bacillus subtilis ThiK (PDB ID 1C3Q), sharing 38% sequence identity, is used as a search model for molecular replacement phasing	Staphylococcus aureus

Crystallization (Comment)

Organism

purified enzyme SaThiM in apoform and in complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues, cpd1 and cpd2, sitting drop vapor diffusion method using 10-20 mg/ml protein in 100 mM Tris, and 150 mM NaCl, pH 8.0, with an equal volume of 0.5 M magnesium formate, microcrystal seeding, with precipitant solution containing 18, 20 or 22% PEG 3350 w/v, 0.2 M magnesium formate, and 5% isopropanol (v/v), against a 0.5 ml reservoir of precipitant, X-ray diffraction structure determination and analysis at 1.62-2.09 A resolution. The homologue structure of Bacillus subtilis ThiK (PDB ID 1C3Q), sharing 38% sequence identity, is used as a search model for molecular replacement phasing

Staphylococcus aureus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.044	-	4-methyl-5-(2-hydroxyethyl)thiazole	pH 7.5, 37°C	Staphylococcus aureus
8.31	-	2-methyl-3-(2-hydroxyethyl)imidazole	pH 7.5, 37°C	Staphylococcus aureus
8.34	-	1,3,5-trimethyl-4-(2-hydroxyethyl)pyrazole	pH 7.5, 37°C	Staphylococcus aureus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	4-methyl-5-(2-hydroxyethyl)thiazole (THZ) forms six direct H-bonds to residues M39, P37, G61, T186 and C190. For the ThiM-THZ complex four Mg2+ ions, essential for the phosphor transfer reaction, are located within four active sites of the two trimers in the asymmetric unit. The Mg2+ ions are coordinated by residues D88, K115 and E120 and one solvent water molecule. No Mg2+ ion is found in the native structure	Staphylococcus aureus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + 4-methyl-5-(2-hydroxyethyl)thiazole	Staphylococcus aureus	-	ADP + 4-methyl-5-(2-phosphooxyethyl)thiazole	-	?
ATP + 4-methyl-5-(2-hydroxyethyl)thiazole	Staphylococcus aureus MRSA252	-	ADP + 4-methyl-5-(2-phosphooxyethyl)thiazole	-	?

Organism

Organism	UniProt	Comment	Textmining
Staphylococcus aureus	Q6GEY3	-	-
Staphylococcus aureus MRSA252	Q6GEY3	-	-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
4.88	-	substrate 4-methyl-5-(2-hydroxyethyl)thiazole, pH 7.5, 37°C	Staphylococcus aureus
7.3	-	substrate 1,3,5-trimethyl-4-(2-hydroxyethyl)pyrazole, pH 7.5, 37°C	Staphylococcus aureus
7.42	-	substrate 2-methyl-3-(2-hydroxyethyl)imidazole, pH 7.5, 37°C	Staphylococcus aureus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + 1,3,5-trimethyl-4-(2-hydroxyethyl)pyrazole	cpd1	Staphylococcus aureus	ADP + 2-(1,3,5-trimethyl-1H-pyrazol-4-yl)ethyl phosphate	-	ir
ATP + 1,3,5-trimethyl-4-(2-hydroxyethyl)pyrazole	cpd1	Staphylococcus aureus MRSA252	ADP + 2-(1,3,5-trimethyl-1H-pyrazol-4-yl)ethyl phosphate	-	ir
ATP + 2-methyl-3-(2-hydroxyethyl)imidazole	cpd2	Staphylococcus aureus	ADP + 2-(2-methyl-1H-imidazol-1-yl)ethyl phosphate	-	ir
ATP + 2-methyl-3-(2-hydroxyethyl)imidazole	cpd2	Staphylococcus aureus MRSA252	ADP + 2-(2-methyl-1H-imidazol-1-yl)ethyl phosphate	-	ir
ATP + 4-methyl-5-(2-hydroxyethyl)thiazole	-	Staphylococcus aureus	ADP + 4-methyl-5-(2-phosphooxyethyl)thiazole	-	?
ATP + 4-methyl-5-(2-hydroxyethyl)thiazole	-	Staphylococcus aureus MRSA252	ADP + 4-methyl-5-(2-phosphooxyethyl)thiazole	-	?
additional information	enzyme-substrate binding structure analysis, overview	Staphylococcus aureus	?	-	-
additional information	enzyme-substrate binding structure analysis, overview	Staphylococcus aureus MRSA252	?	-	-

Subunits

Subunits	Comment	Organism
homotrimer	the quaternary structure of SaThiM is a triangular homotrimer with dimensions of approximately 72 A on a side and 47 A thick with three independent active sites, each located within interface regions between two monomers. Secondary structure of SaThiM consists of approximately 50% alpha-helical and approximately 18% beeta-sheet structural elements, overview	Staphylococcus aureus

Synonyms

Synonyms	Comment	Organism
SaThiM	-	Staphylococcus aureus
ThiM	-	Staphylococcus aureus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Staphylococcus aureus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
2.28	-	4-methyl-5-(2-hydroxyethyl)thiazole	pH 7.5, 37°C	Staphylococcus aureus
3.58	-	1,3,5-trimethyl-4-(2-hydroxyethyl)pyrazole	pH 7.5, 37°C	Staphylococcus aureus
3.63	-	2-methyl-3-(2-hydroxyethyl)imidazole	pH 7.5, 37°C	Staphylococcus aureus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Staphylococcus aureus

General Information

General Information	Comment	Organism
malfunction	thiamin analogous compounds, when introduced into the vitamin B1 biosynthetic pathway and further converted into non-functional cofactors by the bacterium, can function as prodrugs which thus block various cofactor dependent pathways	Staphylococcus aureus
metabolism	enzyme ThiM is involved in the vitamin B1 (thiamin) biosynthetic pathway	Staphylococcus aureus
physiological function	enzyme ThiM is involved in the vitamin B1 (thiamin) biosynthetic pathway. Thiamin in its activated form, thiamin diphosphate, is an essential cofactor for all organisms	Staphylococcus aureus

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.489	-	2-methyl-3-(2-hydroxyethyl)imidazole	pH 7.5, 37°C	Staphylococcus aureus
0.49	-	1,3,5-trimethyl-4-(2-hydroxyethyl)pyrazole	pH 7.5, 37°C	Staphylococcus aureus
51.81	-	4-methyl-5-(2-hydroxyethyl)thiazole	pH 7.5, 37°C	Staphylococcus aureus