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Literature summary extracted from

  • Paul, D.; Chatterjee, A.; Begley, T.P.; Ealick, S.E.
    Domain organization in Candida glabrata THI6, a bifunctional enzyme required for thiamin biosynthesis in eukaryotes (2010), Biochemistry, 49, 9922-9934.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.5.1.3 expressed in Escherichia coli BL21(DE3) cells [Candida] glabrata
2.7.1.50 expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) [Candida] glabrata

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.5.1.3 unliganded and in complex with substrates and products, hanging drop vapor diffusion method, using 0.1 M HEPES (pH 7.5), 0.2-0.25 M MgCl2, and 25-32% PEG400, at 4°C [Candida] glabrata
2.7.1.50 purified recombinant detagged enzyme, in complex with beta,gamma-methyleneadenosine 5'-diphosphate, thiamin phosphate, 4-amino-5-hydroxymethyl-2-trifluoromethylpyrimidine diphosphate, or 4-methyl-5-hydroxyethylthiazole phosphate, hanging-drop vapor diffusion method, mixing of0.001 ml protein solution with 0.001 ml of reservoir solution containing 0.1 M HEPES, pH 7.5, 0.2-0.25 M MgCl2, and 25-32% PEG400, 22°C, 2-3 days, method optimization, X-ray diffraction structure determination and analysis at 2.6-3.3 A resolution, modeling [Candida] glabrata

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.5.1.3 Mg2+ contains Mg2+ [Candida] glabrata
2.7.1.50 Mg2+ required, two ions per enzyme molecule, binding structure involving C467, D340, and E372, overview [Candida] glabrata

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.1.50 ATP + 4-methyl-5-(2-hydroxyethyl)thiazole [Candida] glabrata
-
 ADP + 4-methyl-5-(2-phosphonooxyethyl)thiazole
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.5.1.3 [Candida] glabrata
-
-
-
2.7.1.50 [Candida] glabrata Q6FV03
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.5.1.3 Ni-NTA column chromatography [Candida] glabrata
2.7.1.50 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, cleavage of the tag by TEV protease, and another step of nickel affinity chromatography [Candida] glabrata

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.5.1.3 2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)-thiazole
-
 [Candida] glabrata thiamine phosphate + diphosphate
-
 ?
2.5.1.3 additional information the N-terminal domain of the bifunctional enzyme THI6 catalyzes the ligation of the thiamin thiazole and pyrimidine moieties to form thiamine phosphate, and the C-terminal domain catalyzes the phosphorylation of 4-methyl-5-hydroxyethylthiazole in a salvage pathway. Adenosine diphospho-5beta-ethyl-4-methylthiazole-2-carboxylic acid is not a substrate [Candida] glabrata ?
-
 ?
2.7.1.50 ATP + 4-methyl-5-(2-hydroxyethyl)thiazole
-
 [Candida] glabrata ADP + 4-methyl-5-(2-phosphonooxyethyl)thiazole
-
 ?

Subunits

EC Number Subunits Comment Organism
2.5.1.3 homohexamer
-
 [Candida] glabrata
2.7.1.50 homohexamer the six protomers form a cage-like structure. Each protomer is composed of two domains, which are structurally homologous to their monofunctional bacterial counterparts. Two loop regions not found in the bacterial enzymes provide interactions between the two domains [Candida] glabrata

Synonyms

EC Number Synonyms Comment Organism
2.5.1.3 THI6 bifunctional enzyme [Candida] glabrata
2.5.1.3 thiamin phosphate synthase
-
 [Candida] glabrata
2.5.1.3 TPS
-
 [Candida] glabrata
2.7.1.50 4-methyl-5-hydroxyethylthiazole kinase C-terminal domain of TH16 [Candida] glabrata
2.7.1.50 THI6
-
 [Candida] glabrata
2.7.1.50 ThiM
-
 [Candida] glabrata

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.1.50 ATP
-
 [Candida] glabrata

General Information

EC Number General Information Comment Organism
2.7.1.50 evolution THI6 is a bifunctional enzyme found in the thiamin biosynthetic pathway in eukaryotes. In prokaryotes, thiamin phosphate synthase and 4-methyl-5-hydroxyethylthiazole kinase are separate gene products [Candida] glabrata
2.7.1.50 metabolism THI6 is a bifunctional enzyme of the thiamin biosynthetic pathway [Candida] glabrata
2.7.1.50 physiological function the N-terminal domain of THI6 catalyzes the ligation of the thiamin thiazole and pyrimidine moieties to form thiamin phosphate, and the C-terminal domain catalyzes the phosphorylation of 4-methyl-5-hydroxyethylthiazole in a salvage pathway [Candida] glabrata