Information on EC 4.1.99.12 - 3,4-dihydroxy-2-butanone-4-phosphate synthase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea

EC NUMBER
COMMENTARY hide
4.1.99.12
-
RECOMMENDED NAME
GeneOntology No.
3,4-dihydroxy-2-butanone-4-phosphate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
D-ribulose 5-phosphate = formate + L-3,4-dihydroxybutan-2-one 4-phosphate
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
-
flavin biosynthesis I (bacteria and plants)
-
-
flavin biosynthesis II (archaea)
-
-
flavin biosynthesis III (fungi)
-
-
Metabolic pathways
-
-
Riboflavin metabolism
-
-
flavin biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
D-ribulose 5-phosphate formate-lyase (L-3,4-dihydroxybutan-2-one 4-phosphate-forming)
Requires a divalent cation, preferably Mg2+, for activity [1]. The reaction involves an intramolecular skeletal rearrangement, with the bonds in D-ribulose 5-phosphate that connect C-3 and C-5 to C-4 being broken, C-4 being removed as formate and reconnection of C-3 and C-5 [1]. The phosphorylated four-carbon product (L-3,4-dihydroxybutan-2-one 4-phosphate) is an intermediate in the biosynthesis of riboflavin [1].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene ribA
-
-
Manually annotated by BRENDA team
gene ribB
SwissProt
Manually annotated by BRENDA team
strain TG1
-
-
Manually annotated by BRENDA team
strain W3110
-
-
Manually annotated by BRENDA team
i.e. Pichia guilliermondii
-
-
Manually annotated by BRENDA team
gene RIB3, wild-type strain W303-1A
-
-
Manually annotated by BRENDA team
gene Sp0176
Q97SY7
UniProt
Manually annotated by BRENDA team
gene Sp0176
Q97SY7
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
D-ribulose 5-phosphate
formate + L-3,4-dihydroxy-2-butanone-4-phosphate
show the reaction diagram
D-ribulose 5-phosphate
formate + L-3,4-dihydroxybutan-2-one 4-phosphate
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
D-ribulose 5-phosphate
formate + L-3,4-dihydroxy-2-butanone-4-phosphate
show the reaction diagram
D-ribulose 5-phosphate
formate + L-3,4-dihydroxybutan-2-one 4-phosphate
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
no cofactor required
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
Q97SY7
activates
Fe3+
Q97SY7
the enzyme shows activity in the presence of the trivalent metal ion, Fe3+
Pb2+
Q97SY7
activates
sulfate
-
binding structure at the active site, overview
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.116 - 0.181
D-ribulose 5-phosphate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.109
Q97SY7
substrate D-ribulose 5-phosphate, pH 7.5, 37°C
0.152
-
purified recombinant enzyme
0.174
-
purified enzyme
0.283
purified recombinant enzyme, assay method development, overview
0.332
purified recombinant enzyme
14
-
purified enzyme
additional information
-
development of a spectrophotometric/colorimetric assay method, overview
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 7.7
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
-
enzyme loses activity below pH 5.0 and above pH 9.0
5
-
below pH 5.0 enzyme forms an inactive monomer in solution. The functional activity of Mtb-DHBPS and its dimeric state can be restored by increasing the pH between 6.0 and 9.0
6 - 8
Q97SY7
narrow optimum, no activity below or above, profile overview
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 55
Q97SY7
activity range, profile overview. Highest activity at 33°C, while at 30°C and 37°C, the activity decreases sharply to approximately 15-30% of the activity at 33°C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.8
-
calculated from sequence
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
in the intermembrane space, possibly associated with the outer membrane, minor enzyme portion
Manually annotated by BRENDA team
additional information
-
subcellular localization analysis, overview
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Candida albicans (strain SC5314 / ATCC MYA-2876)
Candida albicans (strain SC5314 / ATCC MYA-2876)
Candida albicans (strain SC5314 / ATCC MYA-2876)
Candida albicans (strain SC5314 / ATCC MYA-2876)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Magnaporthe oryzae (strain 70-15 / ATCC MYA-4617 / FGSC 8958)
Magnaporthe oryzae (strain 70-15 / ATCC MYA-4617 / FGSC 8958)
Magnaporthe oryzae (strain 70-15 / ATCC MYA-4617 / FGSC 8958)
Magnaporthe oryzae (strain 70-15 / ATCC MYA-4617 / FGSC 8958)
Magnaporthe oryzae (strain 70-15 / ATCC MYA-4617 / FGSC 8958)
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
Mycobacterium tuberculosis (strain ATCC 25177 / H37Ra)
Mycobacterium tuberculosis (strain ATCC 25177 / H37Ra)
Mycobacterium tuberculosis (strain ATCC 25177 / H37Ra)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22500
-
x * 28000, SDS-PAGE, x * 22500, about, sequence calculation
22530
2 * 22530, mass spectrometry, 2 * 22658, full-length enzyme, sequence calculation
22658
2 * 22530, mass spectrometry, 2 * 22658, full-length enzyme, sequence calculation
23000
-
2 * 23000, SDS-PAGE, hydrodynamic analysis, NMR structure study, 2 * 23351, mass spectrometry
23177
-
x * 23177, mass spectrometry
23251
x * 23251, recombinant enzyme, mass spectrometry
23351
-
2 * 23000, SDS-PAGE, hydrodynamic analysis, NMR structure study, 2 * 23351, mass spectrometry
24000
-
1 * 24000, SDS-PAGE
25799
-
2 * 25799, recombinant enzyme, mass spectrometry and SDS-PAGE
27860
-
mass spectrometry
28000
-
x * 28000, SDS-PAGE, x * 22500, about, sequence calculation
30000
-
gel filtration
35350
Q97SY7
recombinant enzyme, gel filtration
41000
recombinant enzyme, analytical ultracentrifugation, hydrodynamic studies
42300
x * 42300, recombinant His-tagged N-terminally truncated AtRIBA2
44800
-
recombinant enzyme, analytical ultracentrifugation at 37°C
46300
-
recombinant enzyme, hydrodynamic analysis, NMR structure study, analytical ultracentrifugation at 4°C, overview
47600
x * 47600, recombinant His-tagged N-terminally truncated AtRIBA1
51600
-
recombinant enzyme, analytical ultrafiltration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant apoenzyme in complex with the substrate ribulose 5-phosphate, sitting drop vapour diffusion method, 0.003 ml of 17-34 mg/ml protein in 50 mM Tris-HCl, pH 7.5, is mixed with 0.001 ml of reservoir solution containing 85 mM sodium citrate, pH 5.0, and 17% PEG 8000, with or without 5 mM EDTA, equilibration against 0.3 ml reservoir solution, 0.003 ml of the complex solution is mixed with 0.001 ml of 90 mM Mes/NaOH, pH 6.0, containing 18% PEG 8000, addition of 2 mM D-ribulose 5-phosphate, 20°C, X-ray diffraction structure determination and analysis at 1.6-1.7 A resolution, molecular replacement, modelling
reinterpretation of the space-group symmetry is reported for two crystal structures, PDB codes 1tks and 1tku
purified recombinant enzyme, hanging drop vapour diffusion method at room temperature, 4-5 days, 0.0022 ml of protein solution containing 24 mg/ml protein in 50 mM Tris-HCl pH 7.5, is mixed with 0.0007 ml of precipitating well solution containing 3 M CsCl, 3 M Cs-formate, 20 mM Bis-Tris-propane-NaOH, pH 6.9, or 6 M sodium formate, 25 mM HEPES-NaOH, pH 7.0, labeling with 1.5 mM Au(CN)2, X-ray diffraction structure determination and analysis at 1.4-2.4 A resolution, multiwavelength anomalous diffraction
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purified recombinant enzyme, crystallization of different enzyme complexes: E-SO42-, E-SO42-Mg2+, E-SO42-Mn2+, E-SO42-Mn2+-glycerol, and E-SO42-Zn2+ complexes with X-ray diffraction structure determination and and analysis at resolutions that extend to 1.55 A, 0.98 A, 1.60 A, 1.16 A, and 1.00 A, respectively, divalent cation-free enzyme from 24-30% PEG 5000 monomethyl ether, 0.2 M Li2SO4, and 0.1 M MES-NaOH, pH 6.0-6.5, by the hanging drop vapor diffusion method, to prepare divalent cation-containing crystals, 200 mM MgCl2, 200 mM MnCl2, or 200 mM zinc acetate are added to the crystals for 8-16 h in soaking solutions of the well solutions omitting Li2SO4 and 4% higher in the concentration of PEG 5000 monomethyl ether
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purified recombinnat enzyme divalent cation free, soaked in Zn2+ or soaked in Mg2+, hanging drop vapor diffusion method, room temperature, 0.001 ml of 7 mg/ml protein in 50 mM Tris-HCl, pH 7.5, is mixed with 0.001 ml well solution containing 24-30% PEG monomethyl ether, 0.2 M Li2SO4, and 0.1 M MES-NaOH, pH 6.0-6.5, 1 week-3 months, rectangular plates, X-ray diffraction structure determination and analysis at 1.5 A, 1.0 A, and 1.8 A resolution, respectively
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purified enzyme mutant H147S in complex with substrate ribulose 5-phosphate, monoclinic crystal form, X-ray diffraction structure determination and analysis at 1.55-1.7 A resolution
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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
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three crystal structures of Mtb-DHBPS domain in complex with phosphate and glycerol at pH 6.0, with sulfate at pH 4.0 and with zinc and sulfate at pH 4.0 are determined at 1.8, 2.06 and 2.06 A resolution, respectively
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the crystal structures of Salmonella DHBPS in complex with sulfate, D-ribulose 5-phosphate and sulfate-zinc ion is determined at a resolution of 2.80, 2.52, and 1.86 A, respectively. Analysis of these crystal structures reveals that the acidic loop (residues 34-39) responsible for the acid-base catalysis is disordered in the absence of substrate or metal ion at the active site. Upon binding either substrate or sulfate and metal ions, the acidic loop becomes stabilized, adopts a closed conformation and interacts with the substrate
purified recombinant His-tagged enzyme, hanging-drop vapor diffusion method, mixing of 0.001 ml of 5 mg/ml protein in 5 mM Tris-HCl, pH 8.0, and 50 mM NaCl, with 0.001 ml of reservoir solution containing ammonium sulfate 2.0 M and 0.1 M BisTris pH7.02, X-ray diffraction structure determination and analysis at 2.0 A resolution
Q97SY7
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme by anion and cation exchange chromatography, hydroxyapatite chromatography, and gel filtration, to homogeneity, recombinant enzyme from the overexpressing strain by anion exchange chromatography and gel filtration
native enzyme to homogeneity, by anion and cation exchange chromatography, hydroxyapatite chromatography, and gel filtration
-
recombinant enzyme from Escherichia coli strain BL21(DE3) by anion exchange and hydroxylapatite chromatography, followed by ultrafiltration
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recombinant enzyme from strain Bl21(DE3) by hydrophobic interaction and anion exchange chromatography, and ultrafiltration
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recombinant enzyme from strain M15 by anion exchange chromatography and gel filtration
-
recombinant enzyme, expressed from the synthetic gene in Escherichia coli, to homogeneity by hydrophobic interaction chromatography, ultrafiltration, and gel filtration
recombinant His-tagged DHBPS and GCHII domains from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography and gel filtration
-
recombinant His-tagged N-terminally truncated AtRIBA1 protein from Escherichia coli by nickel affinity chromatography; recombinant His-tagged N-terminally truncated AtRIBA2 protein from Escherichia coli by nickel affinity chromatography
recombinant wild-type and mutant enzymes from Escherichia coli by hydrophobic interaction and hydroxyapatite chromatography, and ultrafiltration
-
recombinant wild-type and mutant soluble His-tagged enzymes from Escherichia coli strain BL21(DE3) by nickel affinity and anion exchange chromatography
Q97SY7
using Ni-NTA chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning by functional complementation of an Escherichia coli DS knockout mutant, expression in Escherichia coli strain BL21(DE3)
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DNA and amino acid sequence determination and analysis, expression of wild-type and mutant enzymes in Escherichia coli strains XL-1 Blue and M15
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expressed in Escherichia coli
expressed in Escherichia coli as a His-tagged fusion protein
expression in Escherichia coli
functional expression of the synthetic gene in Escherichia coli strains XL1-Blue and M15
gene At2g22450, phylogenetic analysis, recombinant expression of His-tagged N-terminally truncated AtRIBA2 protein in Escherichia coli, recombinant expression fo GFP-tagged isozymes AtRIBA1, AtRIBA2, and AtRIBA3 in transgenic Nicotiana benthaminana chloroplasts using Agrobacterium tumefaciens pGV2260 transfection, isozymes AtRIBA2 and AtRIBA3 fail to sufficiently compensate for a lack of RIBA1 expression; gene At5g64300, phylogenetic analysis , recombinant expression of His-tagged N-terminally truncated AtRIBA1 protein in Escherichia coli, recombinant expression of GFP-tagged isozymes AtRIBA1, AtRIBA2, and AtRIBA3 in transgenic Nicotiana benthaminana chloroplasts using Agrobacterium tumefaciens pGV2260 transfection, isozymes AtRIBA2 and AtRIBA3 fail to sufficiently compensate for a lack of RIBA1 expression
gene RIB3, expression of the wild-type gene restores the ability to respire in the aE280/U1 pet mutant A137T of Saccharomyces cerevisiae, which is partially deficient in cytochromes a, a3, and cytochrome b
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gene ribB, DNA determination and analysis, overexpression in strain M15
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gene ribB, overexpression in an rib-defective mutant strain
overexpression in strain 1012 by gene insertion in the sacB locus
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recombinant expression of His-tagged DHBPS and GCHII domains of MtbribA2 in Escherichia coli strain BL21 (DE3)
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recombinant expression of wild-type andn mutant soluble His-tagged enzymes in Escherichia coli strain BL21(DE3)
Q97SY7
subcloning in strain DH5alpha, expression in strain BL21(DE3)
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
increase of 3,4-dihydroxy-2-butanone 4-phosphate synthase upon exposure to Cd2+
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C59A
site-directed mutagenesis, the mutant shows 70% of wild-type enzyme activity
D92A
site-directed mutagenesis, inactive mutant
E166A
site-directed mutagenesis, inactive mutant
Q181R/Q183R
site-directed mutagenesis, construction of a synthetic gene, derived from orf 6.2440, with nucleotide exchanges at positions 414, 426, 477, 480, and 581
Y87A
site-directed mutagenesis, the mutant shows 2% of wild-type enzyme activity
D21E
-
site-directed mutagenesis, inactive mutant
D21N
-
site-directed mutagenesis, inactive mutant
D30E
-
site-directed mutagenesis, inactive mutant
E185D
-
site-directed mutagenesis, inactive mutant
E185X
site-directed mutagenesis, inactive mutant
E26D
-
site-directed mutagenesis, inactive mutant
E26Q
-
site-directed mutagenesis, inactive mutant
E28D
-
site-directed mutagenesis, inactive mutant
H164N
-
site-directed mutagenesis, inactive mutant
E154D
-
DBPS activity is abolished
A137T
-
naturally occuring Rib3 mutant which is partially deficient in cytochromes a, a3, and cytochrome b, the respiratory defect elicited by this mutation cannot be explained by a flavin insufficiency based on the following evidence: 1. growth of the aE280/U1 on respiratory substrates is not rescued by exogenous riboflavin, 2. the levels of flavin nucleotides are not significantly different in the mutant and wild type, phenotype, overview
D32A
Q97SY7
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E163A
Q97SY7
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E30A
Q97SY7
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
H125A
Q97SY7
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
H142A
Q97SY7
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
T143A
Q97SY7
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
T96A
Q97SY7
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
D32A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
-
E163A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
-
E30A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
-
H125A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
-
H142A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
synthesis
-
the enzyme is essential for industrial riboflavin production by Bacillus subtilis overproducing strains, overview
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