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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 PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.5.1.3 expressed in Escherichia coli BL21(DE3) cells Nakaseomyces glabratus
2.7.1.50 expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Nakaseomyces glabratus

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 Nakaseomyces glabratus
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 Nakaseomyces glabratus

Metals/Ions

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

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 Nakaseomyces glabratus
-
 ADP + 4-methyl-5-(2-phosphonooxyethyl)thiazole
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.5.1.3 Nakaseomyces glabratus
-
-
-
2.7.1.50 Nakaseomyces glabratus Q6FV03
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.5.1.3 Ni-NTA column chromatography Nakaseomyces glabratus
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 Nakaseomyces glabratus

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
-
 Nakaseomyces glabratus 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 Nakaseomyces glabratus ?
-
 ?
2.7.1.50 ATP + 4-methyl-5-(2-hydroxyethyl)thiazole
-
 Nakaseomyces glabratus ADP + 4-methyl-5-(2-phosphonooxyethyl)thiazole
-
 ?

Subunits

EC Number Subunits Comment Organism
2.5.1.3 homohexamer
-
 Nakaseomyces glabratus
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 Nakaseomyces glabratus

Synonyms

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

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.1.50 ATP
-
 Nakaseomyces glabratus

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 Nakaseomyces glabratus
2.7.1.50 metabolism THI6 is a bifunctional enzyme of the thiamin biosynthetic pathway Nakaseomyces glabratus
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 Nakaseomyces glabratus