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Literature summary extracted from

  • Singh, M.; Kumar, P.; Yadav, S.; Gautam, R.; Sharma, N.; Karthikeyan, S.
    The crystal structure reveals the molecular mechanism of bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II (Rv1415) from Mycobacterium tuberculosis (2013), Acta Crystallogr. Sect. D, 69, 1633-1644.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.4.25 expressed in Escherichia coli BL21(DE3) cells Mycobacterium tuberculosis
4.1.99.12 recombinant expression of His-tagged DHBPS and GCHII domains of MtbribA2 in Escherichia coli strain BL21 (DE3) Mycobacterium tuberculosis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.5.4.25 sitting drop vapor diffusion method, using 0.1 M Na HEPES pH 7.5, 15% (w/v) PEG 8000 Mycobacterium tuberculosis
4.1.99.12 purified recombinant bifunctional enzyme, sitting drop vapour diffusion method, mixing of 0.001 ml of 20-25 mg/ml protein in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, with 0.001 ml of reservoir solutioncontaining 0.1 M Na HEPES pH 7.5, 15%(w/v) PEG 8000, equilibration against 0.04 ml reservoir solution, 20°C, 2-4 days, X-ray diffraction structure determination and analysis at 3.0 A resolution Mycobacterium tuberculosis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.4.25 0.1267
-
GTP full-length bifunctional enzyme ribA2, at pH 8.0 and 37°C Mycobacterium tuberculosis
3.5.4.25 0.1505
-
GTP GCHII-domain of enzyme ribA2, at pH 8.0 and 37°C Mycobacterium tuberculosis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.5.4.25 Mg2+ required Mycobacterium tuberculosis
3.5.4.25 Zn2+ contains zinc Mycobacterium tuberculosis
4.1.99.12 Mg2+ essential for catalytic activity and in stabilization of the substrate Mycobacterium tuberculosis
4.1.99.12 additional information the GTP cyclohydrolase II subunit has also bound Zn2+ ions Mycobacterium tuberculosis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.5.4.25 48163
-
2 * 48163, MALDI-TOF mass spectrometry Mycobacterium tuberculosis
3.5.4.25 90000
-
gel filtration Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.4.25 GTP + H2O Mycobacterium tuberculosis
-
formate + 2,5-diamino-6-hydroxy-4-(5-phospho-D-ribosylamino)pyrimidine + diphosphate
-
?
3.5.4.25 GTP + H2O Mycobacterium tuberculosis H37Rv
-
formate + 2,5-diamino-6-hydroxy-4-(5-phospho-D-ribosylamino)pyrimidine + diphosphate
-
?
4.1.99.12 D-ribulose 5-phosphate Mycobacterium tuberculosis
-
formate + L-3,4-dihydroxybutan-2-one 4-phosphate
-
?
4.1.99.12 D-ribulose 5-phosphate Mycobacterium tuberculosis H37Ra / ATCC 25177
-
formate + L-3,4-dihydroxybutan-2-one 4-phosphate
-
?

Organism

EC Number Organism UniProt Comment Textmining
3.5.4.25 Mycobacterium tuberculosis P9WHF1
-
-
3.5.4.25 Mycobacterium tuberculosis H37Rv P9WHF1
-
-
4.1.99.12 Mycobacterium tuberculosis A5U2B7
-
-
4.1.99.12 Mycobacterium tuberculosis H37Ra / ATCC 25177 A5U2B7
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.4.25 ammonium sulfate precipitation, Ni-NTA column chromatography, and Sephacryl S-200 gel filtration Mycobacterium tuberculosis
4.1.99.12 recombinant His-tagged DHBPS and GCHII domains from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography and gel filtration Mycobacterium tuberculosis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.4.25 GTP + H2O
-
Mycobacterium tuberculosis formate + 2,5-diamino-6-hydroxy-4-(5-phospho-D-ribosylamino)pyrimidine + diphosphate
-
?
3.5.4.25 GTP + H2O
-
Mycobacterium tuberculosis H37Rv formate + 2,5-diamino-6-hydroxy-4-(5-phospho-D-ribosylamino)pyrimidine + diphosphate
-
?
4.1.99.12 D-ribulose 5-phosphate
-
Mycobacterium tuberculosis formate + L-3,4-dihydroxybutan-2-one 4-phosphate
-
?
4.1.99.12 D-ribulose 5-phosphate
-
Mycobacterium tuberculosis H37Ra / ATCC 25177 formate + L-3,4-dihydroxybutan-2-one 4-phosphate
-
?

Subunits

EC Number Subunits Comment Organism
3.5.4.25 homodimer 2 * 48163, MALDI-TOF mass spectrometry Mycobacterium tuberculosis
4.1.99.12 dimer two conformationally different molecules of Mtb-ribA2 in the asymmetric unit that form a dimer via their GCHII domains. DHBPS and GCHII functional homodimers form a long helical-like oligomer, but the enzyme exists as a dimer in solution, isolated subunit Mtb-GCHII is a dimer while Mtb-DHBPS exists as amonomer in solution. The N-terminal DHBPS domain of Mtb-ribA2 shows an alpha + beta fold consisting of a central eight-stranded beta-sheet (beta1-beta8) surrounded by seven helices (alpha1-7alpha7) Mycobacterium tuberculosis

Synonyms

EC Number Synonyms Comment Organism
3.5.4.25 GCHII
-
Mycobacterium tuberculosis
3.5.4.25 ribA2 bifunctional enzyme with 3,4-dihydroxy-2-butanone 4-phosphate synthase and GTP cyclohydrolase II activities Mycobacterium tuberculosis
4.1.99.12 DHBPS
-
Mycobacterium tuberculosis
4.1.99.12 DHBPS/GCHII
-
Mycobacterium tuberculosis
4.1.99.12 Mtb-ribA2
-
Mycobacterium tuberculosis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.4.25 0.0033
-
GTP full-length bifunctional enzyme ribA2, at pH 8.0 and 37°C Mycobacterium tuberculosis
3.5.4.25 0.005
-
GTP GCHII-domain of enzyme ribA2, at pH 8.0 and 37°C Mycobacterium tuberculosis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.1.99.12 8
-
assay at Mycobacterium tuberculosis

General Information

EC Number General Information Comment Organism
4.1.99.12 additional information an essential bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II (DHBPS/GCHII) enzyme from Mycobacterium tuberculosis, Mtb-ribA2. The enzyme is composed of two conformationally different molecules of Mtb-ribA2 in the asymmetric unit that form a dimer via their GCHII domains. DHBPS and GCHII functional homodimers form a long helical-like oligomer, but the enzyme exists as a dimer in solution. The DHBPS subunit possesses an active-site loop1, a substrate channelling loop2, and a pH-sensitive loop3, structure comparisons, overview Mycobacterium tuberculosis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.5.4.25 1.4
-
GTP full-length bifunctional enzyme ribA2, at pH 8.0 and 37°C Mycobacterium tuberculosis
3.5.4.25 2.1
-
GTP GCHII-domain of enzyme ribA2, at pH 8.0 and 37°C Mycobacterium tuberculosis