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Literature summary for 3.5.4.25 extracted from

  • Kaiser, J.; Schramek, N.; Eberhardt, S.; Puttmer, S.; Schuster, M.; Bacher, A.
    Biosynthesis of vitamin B2. An essential zinc ion at the catalytic site of GTP cyclohydrolase II (2002), Eur. J. Biochem., 269, 5264-5270.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutant enzymes Escherichia coli

Protein Variants

Protein Variants Comment Organism
C54S site-directed mutagenesis, mutation results in proteins devoid of bound zinc and unable to release formate from the imidazole ring of GTP or from the intermediate analogue 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, however, the mutant enzyme is still capable to release diphosphate from GTP and from the formamide-type intermediate analogue Escherichia coli
C65S site-directed mutagenesis, mutation results in proteins devoid of bound zinc and unable to release formate from the imidazole ring of GTP or from the intermediate analogue 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, however, the mutant enzyme is still capable to release diphosphate from GTP and from the formamide-type intermediate analogue Escherichia coli
C67S site-directed mutagenesis, mutation results in proteins devoid of bound zinc and unable to release formate from the imidazole ring of GTP or from the intermediate analogue 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-triphosphate, however, the mutant enzyme is still capable to release diphosphate from GTP and from the formamide-type intermediate analogue Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Zn2+ wild-type enzyme contains 0.71 mol Zn2+ per mol of subunit, while the content in the mutant enzymes is reduced to below 0.1 mol Zn2+ per mol of subunit, essential for activity, bound to the active site, required for ring opening and formate release with zinc acting as a Lewis acid, which activates the 2 water molecules involved in the sequential hydrolysis of 2 carbon-nitrogen bonds Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
GTP + H2O Escherichia coli first committed step in the biosynthesis of riboflavin, overview formate + 2,5-diamino-6-hydroxy-4-(5-phosphoribosylamino)pyrimidine + diphosphate
-
?

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
-
-

Reaction

Reaction Comment Organism Reaction ID
GTP + 4 H2O = formate + 2,5-diamino-6-hydroxy-4-(5-phospho-D-ribosylamino)pyrimidine + 2 phosphate ordered reaction mechanism, in which the hydrolytic release of diphosphate preceeds the hydrolytic attack of the imidazole ring, ring opening and formate release are dependent on zinc bound to the active site, overview Escherichia coli

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
activity of the mutant enzyme C54S, C65S, and C67S with the different substrates, overview, best substrate is GTP being cleaved to GMP Escherichia coli
0.002
-
purified recombinant mutant C65S, substrate GTP cleaved to GMP Escherichia coli
0.003
-
purified recombinant mutant C67S, substrate GTP cleaved to GMP Escherichia coli
0.01
-
purified recombinant mutant C54S, substrate GTP cleaved to GMP Escherichia coli
0.015
-
purified recombinant wild-type enzyme, substrate 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate cleaved to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate Escherichia coli
0.016
-
purified recombinant wild-type enzyme, substrate GTP cleaved to GMP Escherichia coli
0.025
-
purified recombinant wild-type enzyme, substrate dGTP cleaved to dGMP Escherichia coli
0.038
-
purified recombinant wild-type enzyme, substrate dGTP cleaved to 2,5-diamino-6-hydroxy-4-(5-phospho-deoxyribosylamino)pyrimidine Escherichia coli
0.122
-
purified recombinant wild-type enzyme, substrate 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate cleaved to 2,5-diamino-6-hydroxy-4-(5-phosphoribosylamino)pyrimidine Escherichia coli
0.182
-
purified recombinant wild-type enzyme, substrate GTP cleaved to 2,5-diamino-6-hydroxy-4-(5-phosphoribosylamino)pyrimidine Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate + 2 H2O formamide-type intermediate analogue Escherichia coli formate + 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate + diphosphate
-
?
2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate + H2O formamide-type intermediate analogue Escherichia coli 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate + diphosphate
-
?
dGTP + H2O
-
Escherichia coli formate + 2,5-diamino-6-hydroxy-4-(5-phospho-deoxyribosylamino)pyrimidine + diphosphate
-
?
GTP + H2O
-
Escherichia coli formate + 2,5-diamino-6-hydroxy-4-(5-phosphoribosylamino)pyrimidine + diphosphate
-
?
GTP + H2O first committed step in the biosynthesis of riboflavin, overview Escherichia coli formate + 2,5-diamino-6-hydroxy-4-(5-phosphoribosylamino)pyrimidine + diphosphate
-
?

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Escherichia coli