Literature summary for 4.1.99.12 extracted from

Islam, Z.; Kumar, A.; Singh, S.; Salmon, L.; Karthikeyan, S.
Structural basis for competitive inhibition of 3,4-dihydroxy-2-butanone-4-phosphate synthase from Vibrio cholerae (2015), J. Biol. Chem., 290, 11293-11308 .

Search for more literature for 4.1.99.12

Cloned(Commentary)

Cloned (Comment)	Organism
gene ribB, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Vibrio cholerae serotype O1

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme in apoform or in complex with inhibitor 4-phospho-D-erythronohydroxamic acid, or substrate and substrate plus metal ions, sitting drop vapor diffusion method, mixing of 0.001 ml of 20 mg/ml protein in 25 mM Tris-HCl, pH 8.0, with 0.01 ml crystallization solution, containing for the apo form enzyme 0.2 M Na2HPO4, pH 9.1, and 20% PEG 3350, for phosphate-bound enzyme 0.2 M NaH2PO4, pH 4.5 and 20% PEG 3350, the Ru5P and 4PEH complex crystals are obtained by mixing vDHBPS with 10 mM Ru5P or 4PEH and incubation for 30 min at 20°C, grown in 0.2 M Na2HPO4, pH 9.1 and 20% PEG3350, the enzyme-Ru5P-Zn2+ and enzyme-4PEH-Zn2+ complex crystals are obtained by soaking complex crystals in 100 mM ZnCl2 for 12 h at 20°C, X-ray diffraction structure determination and analysis at 1.96 A, 1.86 A, 1.59 A, and 2.04 A resolution, respectively, modelling	Vibrio cholerae serotype O1

Crystallization (Comment)

Organism

purified enzyme in apoform or in complex with inhibitor 4-phospho-D-erythronohydroxamic acid, or substrate and substrate plus metal ions, sitting drop vapor diffusion method, mixing of 0.001 ml of 20 mg/ml protein in 25 mM Tris-HCl, pH 8.0, with 0.01 ml crystallization solution, containing for the apo form enzyme 0.2 M Na2HPO4, pH 9.1, and 20% PEG 3350, for phosphate-bound enzyme 0.2 M NaH2PO4, pH 4.5 and 20% PEG 3350, the Ru5P and 4PEH complex crystals are obtained by mixing vDHBPS with 10 mM Ru5P or 4PEH and incubation for 30 min at 20°C, grown in 0.2 M Na2HPO4, pH 9.1 and 20% PEG3350, the enzyme-Ru5P-Zn2+ and enzyme-4PEH-Zn2+ complex crystals are obtained by soaking complex crystals in 100 mM ZnCl2 for 12 h at 20°C, X-ray diffraction structure determination and analysis at 1.96 A, 1.86 A, 1.59 A, and 2.04 A resolution, respectively, modelling

Vibrio cholerae serotype O1

Protein Variants

Protein Variants	Comment	Organism
E39A	site-directed mutagenesis, inactive mutant	Vibrio cholerae serotype O1
E41A	site-directed mutagenesis, the mutant is almost inactive	Vibrio cholerae serotype O1
F139A	site-directed mutagenesis, the mutant shows moderately reduced activity compared to the wild-type enzyme	Vibrio cholerae serotype O1
H154A	site-directed mutagenesis, inactive mutant	Vibrio cholerae serotype O1
M84A	site-directed mutagenesis, the mutant shows moderately reduced activity compared to the wild-type enzyme	Vibrio cholerae serotype O1
N89A	site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme	Vibrio cholerae serotype O1
R61A	site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme	Vibrio cholerae serotype O1
S64A	site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme	Vibrio cholerae serotype O1
V98A	site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme	Vibrio cholerae serotype O1

Inhibitors

Inhibitors	Comment	Organism	Structure
4-phospho-D-erythronohydroxamic acid	4PEH, structure analogue of D-ribulose 5-phosphate, competitive inhibitor, 4PEH inhibits the catalytic activity of DHBPS as it is unable to form a proposed intermediate that is crucial for DHBPS activity	Vibrio cholerae serotype O1

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Vibrio cholerae serotype O1
Zn2+	required, binding structure analysis	Vibrio cholerae serotype O1

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
D-ribulose 5-phosphate	Vibrio cholerae serotype O1	-	formate + L-3,4-dihydroxybutan-2-one 4-phosphate	-	?
D-ribulose 5-phosphate	Vibrio cholerae serotype O1 ATCC 39315 / El Tor Inaba N16961	-	formate + L-3,4-dihydroxybutan-2-one 4-phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
no activity in Homo sapiens	-	-	-
Vibrio cholerae serotype O1	Q9KKP2	-	-
Vibrio cholerae serotype O1 ATCC 39315 / El Tor Inaba N16961	Q9KKP2	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, and gel filtration, followed by ultrafiltration	Vibrio cholerae serotype O1

Reaction

Reaction	Comment	Organism	Reaction ID
D-ribulose 5-phosphate = formate + L-3,4-dihydroxybutan-2-one 4-phosphate	reaction mechanism, detailed overview	Vibrio cholerae serotype O1	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
D-ribulose 5-phosphate	-	Vibrio cholerae serotype O1	formate + L-3,4-dihydroxybutan-2-one 4-phosphate	-	?
D-ribulose 5-phosphate	-	Vibrio cholerae serotype O1 ATCC 39315 / El Tor Inaba N16961	formate + L-3,4-dihydroxybutan-2-one 4-phosphate	-	?
additional information	substrate binding structures, overview	Vibrio cholerae serotype O1	?	-	?
additional information	substrate binding structures, overview	Vibrio cholerae serotype O1 ATCC 39315 / El Tor Inaba N16961	?	-	?

Subunits

Subunits	Comment	Organism
dimer	the hydrophobic residues at the dimeric interface play a role in dimer formation, whereas some of the active site residues at the dimeric interface play a role in the catalytic activity of vDHBPS	Vibrio cholerae serotype O1
More	DHBPS enzyme structure comparisons	Vibrio cholerae serotype O1

Synonyms

Synonyms	Comment	Organism
3,4-dihydroxy-2-butanone-4-phosphate synthase	-	Vibrio cholerae serotype O1
DHBPS	-	Vibrio cholerae serotype O1
ribB	-	Vibrio cholerae serotype O1
vDHBPS	-	Vibrio cholerae serotype O1
vribB	-	Vibrio cholerae serotype O1

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Vibrio cholerae serotype O1

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Vibrio cholerae serotype O1

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.1	-	4-phospho-D-erythronohydroxamic acid	pH 7.5, 37°C, recombinant His-tagged wild-type enzyme	Vibrio cholerae serotype O1

General Information

General Information	Comment	Organism
additional information	DHBPS enzyme structure comparisons, active site architecture of enzyme vDHBPS	Vibrio cholerae serotype O1
physiological function	enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes one of the two committed steps in the riboflavin pathway and converts D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone 4-phosphate and formate	Vibrio cholerae serotype O1