Information on EC 2.7.6.3 - 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase

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

EC NUMBER
COMMENTARY hide
2.7.6.3
-
RECOMMENDED NAME
GeneOntology No.
2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + 6-hydroxymethyl-7,8-dihydropterin = AMP + 6-hydroxymethyl-7,8-dihydropterin diphosphate
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
diphosphate transfer
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
6-hydroxymethyl-dihydropterin diphosphate biosynthesis
-
-
6-hydroxymethyl-dihydropterin diphosphate biosynthesis I
-
-
6-hydroxymethyl-dihydropterin diphosphate biosynthesis II (archaea)
-
-
6-hydroxymethyl-dihydropterin diphosphate biosynthesis III (Chlamydia)
-
-
Folate biosynthesis
-
-
Metabolic pathways
-
-
tetrahydromethanopterin biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
ATP:6-hydroxymethyl-7,8-dihydropterin 6'-diphosphotransferase
Binds 2 Mg2+ ions that are essential for activity [4]. The enzyme participates in the biosynthetic pathways for folate (in bacteria, plants and fungi) and methanopterin (in archaea). The enzyme exists in varying types of multifunctional proteins in different organisms. The enzyme from the bacterium Streptococcus pneumoniae also harbours the activity of EC 4.1.2.25, dihydroneopterin aldolase [4], the enzyme from the plant Arabidopsis thaliana harbours the activity of EC 2.5.1.15, dihydropteroate synthase [7], while the enzyme from yeast Saccharomyces cerevisiae is trifunctional with both of the two above mentioned activities [6].
CAS REGISTRY NUMBER
COMMENTARY hide
37278-23-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
Q2A2W3
UniProt
Manually annotated by BRENDA team
-
Q2A2W3
UniProt
Manually annotated by BRENDA team
Brookhaven Protein Data Bank: 1cbk
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
-
6-hydroxymethyl-7,8-dihydropterin pyrophosphokinae is a committed enzyme in the folate pathway of Streptococcus aureus playing an integral role in the biosynthesis of tetrahydrofolate
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
6-hydroxymethyl-7,8-dihydropterin + ATP
6-hydroxymethyl-7,8-dihydropterin diphosphate + AMP
show the reaction diagram
6-hydroxymethyl-7,8-dihydropterin + MgATP2-
6-hydroxymethyl-7,8-dihydropterin diphosphate + MgAMP
show the reaction diagram
-
biosynthesis of folate cofactors
-
-
?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + 2-amino-7,8-dihydro-4-hydroxy-6-(diphosphooxymethyl)pteridine
show the reaction diagram
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 6-hydroxymethyl-7,8-dihydropteridine diphosphate
show the reaction diagram
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 7,8-dihydro-6-(diphosphooxymethyl)pteridine
show the reaction diagram
-
-
-
-
?
ATP + 6-hydroxymethyl-7,8-dihydropterin
AMP + (7,8-dihydropterin-6-yl)methyl diphosphate
show the reaction diagram
dATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
dAMP + 2-amino-7,8-dihydro-4-hydroxy-6-(diphosphooxymethyl)pteridine
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
6-hydroxymethyl-7,8-dihydropterin + ATP
6-hydroxymethyl-7,8-dihydropterin diphosphate + AMP
show the reaction diagram
6-hydroxymethyl-7,8-dihydropterin + MgATP2-
6-hydroxymethyl-7,8-dihydropterin diphosphate + MgAMP
show the reaction diagram
-
biosynthesis of folate cofactors
-
-
?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + 2-amino-7,8-dihydro-4-hydroxy-6-(diphosphooxymethyl)pteridine
show the reaction diagram
-
key step in biosynthesis of folic acid
-
-
?
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 6-hydroxymethyl-7,8-dihydropteridine diphosphate
show the reaction diagram
ATP + 6-hydroxymethyl-7,8-dihydropterin
AMP + (7,8-dihydropterin-6-yl)methyl diphosphate
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
less effective than Mn2+ or Mg2+ in activation
KCl
-
activation at 0.4 M, inhibition at higher concentration
Mn2+
-
can replace Mg2+ with a 10 mM optimum
NaCl
-
activation at 0.2 M, inhibition at higher concentration
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-Amino-4-hydroxy-6-carboxydihydropteridine
-
competitive with 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine
2-amino-6-[(2-[4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-ylmethylsulfanyl]-piperidin-1-yl]-ethylamino)-methyl]-3H-pteridin-4-one
-
-
2-amino-6-[(2-[4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-ylmethylsulfanyl]-piperidin-1-yl]-ethylamino)-methyl]-7,7-dimethyl-7,8-dihydro-3H-pteridin-4-one
-
-
2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic acid (2-[4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethylsulfanyl]-piperidin-1-yl]-ethyl)-amide
-
-
2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridine-6-carboxylic acid (2-[2-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethanesulfonyl]-ethylcarbamoyl]-ethyl)-amide
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about 45% residual activity at 0.01 mM, about 30% residual activity at 0.02 mM, about 15% residual activity at 0.05 mM, almost complete inhibition at 0.1 mM
5'-S-[1-(2-{[(2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridin-6-yl)carbonyl]amino}ethyl)piperidin-4-yl]-5'-thioadenosine
-
-
6-hydroxymethyl-7,7-dimethyl-7,8-dihydropterin
-
-
6-hydroxymethyl-7,8-dihydropterin diphosphate
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competitive inhibitor of ATP, mixed type inhibitor of 6-hydroxymethyl-7,8-dihydropteridine
6-hydroxymethyl-7-methyl-7-phenethyl-7,8-dihydropterin
-
-
alpha,beta-methyleneadenosine triphosphate
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competitive with respect to ATP
AMP
-
poor inhibitor
Guanidine-HCl
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0.25 M, 50% inhibitioin
KCl
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activation at 0.4 M, inhibition at higher concentration
NaCl
-
activation at 0.2 M, inhibition at higher concentration
P1-(6-hydroxymethylpterin)-P2-(5'-adenosyl)diphosphate
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P1-(6-hydroxymethylpterin)-P3-(5'-adenosyl)triphosphate
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P1-(6-hydroxymethylpterin)-P4-(5'-adenosyl)tetraphosphate
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sulfachloropyridazine
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sulfadoxine
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competitive, moderately potent
sulfathiazole
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competitive
Urea
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0.9 M, 50% inhibition
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01 - 0.015
2-amino-4-hydroxy-6-hydroxymethyldihydropteridine
0.001 - 0.0036
6-hydroxymethyl-7,8-dihydropteridine
0.00039
6-hydroxymethyl-7,8-dihydropterin
-
0.011 - 0.07
ATP
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
Escherichia coli
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-
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.043
2-Amino-4-hydroxy-6-carboxydihydropteridine
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pH 8.5, 37C
0.005 - 0.013
6-hydroxymethyl-7,8-dihydropterin diphosphate
0.00031
alpha,beta-methyleneadenosine triphosphate
-
-
0.4 - 0.7
AMP
0.011
sulfadoxine
-
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1
2-amino-6-[(2-[4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-ylmethylsulfanyl]-piperidin-1-yl]-ethylamino)-methyl]-3H-pteridin-4-one
Escherichia coli
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IC50 above 0.1 mM, in 100 mM Tris, pH 8.3, temperature not specified in the publication
0.00316
2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic acid (2-[4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethylsulfanyl]-piperidin-1-yl]-ethyl)-amide
Escherichia coli
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in 100 mM Tris, pH 8.3, temperature not specified in the publication
0.00953
2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridine-6-carboxylic acid (2-[2-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethanesulfonyl]-ethylcarbamoyl]-ethyl)-amide
Escherichia coli
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in 100 mM Tris, pH 8.3, at 23C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5 - 9
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Tris buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 10.8
-
active between pH 7.5 and pH 10.8
8.5 - 10.5
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pH 8.5: about 40% of maximal activity, pH 10.5: about 90% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 45
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30C: about 40% of maximal activity, 37-45C: optimum
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.55 - 4.65
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isoelectric focusing
7.1
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calulation from amino acid sequence
9.1
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isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
developing, isozyme cytHPPK/DHPS gene is exclusively expressed in developing seeds, histochemical analysis of a transgenic cytHPPK/DHPS promoter-GUS line
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
isozyme cytHPPK/DHPS lacking a potential transit peptide
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
Streptococcus pneumoniae (strain ATCC BAA-255 / R6)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20000
-
gel filtration, equilibrium sedimentation, quasi-elastic light scattering
25000
-
gel filtration
69000
-
calculation from multifunctional folic acid synthesis fas gene that encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase
150000
-
gel filtration
190000
-
gel filtration
330000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
Arg79 and Gln88 may help to stabilize the homodimer. The long side chain of Arg79 in YpHPPK forms many hydrophobic interactions with Trp90 and Gln49 from the partner subunit and a weak hydrogen bond with the side chain of Gln49, which may help to stabilize the dimeric molecule
monomer
tetramer
-
4 * 83000, SDS-PAGE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
apo W89A and its ternary complex with Mg-alpha,beta-methyleneadenosine triphosphate and 6-hydroxymethyl-7,8-dihydropterin are crystallized at 19C using the hanging-drop vapor-diffusion technique. The structure of the ternary complex is determined at 1.25 A resolution
-
complexed with inhibitor 2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridine-6-carboxylic acid (2-[2-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethanesulfonyl]-ethylcarbamoyl]-ethyl)-amide, sitting drop vapor diffusion method, using 25% (w/v) PEG 3350 and 0.2 M NaCl in 0.1 M HEPES, pH 7.5
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hanging-drop vapor-diffusion method. At 0.89-A resolution, two distinct conformations are observed for each of the two residues in the crystal structure of the wild-type enzyme in complex with two 6-hydroxymethyl-7,8-dihydropterin variants, two Mg2+ ions, and an ATP analogue. 1. Complex of wild-type enzyme with 6-hydroxymethylpterin, 6-carboxypterin and alpha,beta-methyleneadenosine 5'-triphosphate, 2. complex of mutant enzyme R82A with 6-hydroxymethyl-7,8-dihydropterin and alpha,beta-methyleneadenosine 5'-triphosphate, 3. complex of mutant enzyme R92A with 6-hydroxymethyl-7,8-dihydropterin and alpha,beta-methyleneadenosine 5'-triphosphate, 4. matant apoenzyme of R82A, 5. mutant apoenzyme of R92A, 6. mutant enzyme R92A in complex with Mg2+
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in complex with 2-amino-6-[(2-{4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-ylmethylsulfanyl]-piperidin-1-yl}-ethylamino)-methyl]-3H-pteridin-4-one, 2-amino-6-[(2-{4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-ylmethylsulfanyl]-piperidin-1-yl}-ethylamino)-methyl]-7,7-dimethyl-7,8-dihydro-3H pteridin-4-one, or 2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic acid (2-{4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethylsulfanyl]-piperidin-1-yl}-ethyl)-amide, sitting drop vapor diffusion method, using 20% or 25% (w/v) PEG 3350 as precipitant, at 19C
-
V83Gdel84-89 and its complex with alpha,beta--methyleneadenosine triphosphate and 6-hydroxymethyl-7,8-dihydropterin are crystallized at 19C using the hanging-drop vapor-diffusion technique
-
apoenzyme and in complex with substrate 6-hydroxymethyl-7,8-dihydropteridine or 2-(7-amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydorpyrimido[4,5-c]pyridazin-3-yl)propanoic acid, sitting drop vapor diffusion method, using 90 mM Tris (pH 8.0), 190 mM sodium acetate, 24% (w/v) polyethylene glycol (PEG) 4000, and 17% (v/v) glycerol, at 18C
Q2A2W3
a complex of the purified protein with a substrate analog is crystallized and its structure is solved by multiple anomalous dispersion using phase information obtained from a single crystal of selenomethionine-labeled protein
-
crystallization of a complex of the purified bifunctional polypeptide with a pterin monophosphate substrate analogue, structure solved by molecular replacement and refined to 2.3 A resolution. Three-dimensional structure in complex with the oxidized substrate analogue 6-hydroxy-methyl-pterin monophosphate reveals how the HPPK and DHPS functional domains associate at both the ternary and quaternary levels
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sitting drop vapor diffusion method, using 1.08 M sodium malonate pH 7, 0.09 M bis-Tris pH 6.5, 0.175 M sodium formate, and 0.01 M sodium acetate pH 4.6
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hanging-drop method
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purified recombinant HPPK in complex with 6-hydroxymethyl-7,8-dihydropterin and an ATP analogue AMPCPP, 8.0 mg/ml HPPK in 15 mM HP, 25 mM AMPCPP, 50 mM MgCl2 and 10 mM Tris-HCl, pH 8.0 s mixed with well solution containing 0.18 M ammonium acetate, 30% w/v PEG 4000, 20 mM imidazole, and 0.1 M sodium acetate, pH 4.6, 1-3 weeks, X-ray diffraction structure determination and analysis, molecular replacement
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
-
complete inactivation
100
-
60 min, 75% loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C or -80C for 3 months in 20 mM Tris-HCl, pH 8.0 buffer containing 10 or 20% glycerol, no loss of activity. In buffer without glycerol, 30 and 50% of the enzyme activity are lost when stored for 6 months at -80C and -20C, respectively
-
-20C, enzyme loses activity over a period of months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
HPPK-glutathione S-transferase fusion proteins
-
HPPK-GST fusion proteins
-
Ni-NTA column chromatography and Superdex 75 gel filtration
recombinant bifunctional protein 6-hydroxymethyl-7,8-dihydroxypterin pyrophosphokinase/7,8-dihydropteroate synthase is involved in tetrahydrofolate
-
recombinant His6-tagged enzyme from Escherichia coli by metal affinity chromatography and ion exchange chromatography, cleavage of the tag by thrombin
recombinant N-terminally His6-tagged isozyme cytHPPK/DHPS from Escherichia coli strains DH-5alpha and BL-21AI
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
bifunctional protein 6-hydroxymethyl-7,8-dihydroxypterin pyrophosphokinase/7,8-dihydropteroate synthase is involved in tetrahydrofolate expressed in Escherichia coli
-
expressed in Escherichia coli BL21 cells
Q2A2W3
expressed in Escherichia coli BL21(DE3) cells
-
expression in Escherichia coli
expression in Escherichia coli and Saccharomyces cerevisiae; isozyme cytHPPK/DHPS DNA and amino acid sequence determination and analysis, and phylogenetic analysis, transient expression of GFP-tagged isozyme cytHPPK/DHPS in the cytosol of Arabidopsis thaliana protoplasts using transfection via Agrobacterium tumefaciens strain GV3101/pMP90, functional complementation of a Saccharomyces cerevisiae mutant, that lacks the enzyme activity, by expression of isozyme cytHPPK/DHPS, expression and subcloning of N-terminally His6-tagged isozyme cytHPPK/DHPS in Escherichia coli strains DH-5alpha and BL-21AI, co-expression with GroESL chaperones
expression in Escherichia coli; expression of His6-tagged enzyme in Escherichia coli
expression of His6-tagged maltose-binding-protein fusion HPPK in Escherichia coli strain BL21(DE3)
expression of the bifunctional 6-hydroxymethyl-7,8-dihydropterin diphosphokinase/dihydropteroate synthase in Escherichia coli
-
hydroxymethyldihydropterin diphosphokinase from Plasmodium falciparum complements a folK-knockout mutant in Escherichia coli when expressed as a separate polypeptide detached from dihydropteroate synthase. Hydroxymethyldihydropterin diphosphokinase part of the bifunctional protein can function by itself but that a larger part of the polypeptide is needed to ensure full functionality
-
multifunctional folic acid synthesis fas gene that encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase, in cultured Spodoptera frugiperda SF9 insect cells
-
the hydroxymethyldihydropterin pyrophosphokinase domain of the multifunctional folic acid synthesis Fas protein expressed as an independent enzyme in Escherichia coli, high level expression in inclusion bodies using an inducible tac promoter expression system
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R82A
-
mutation causes a decrease in the rate constant for the chemical step by a factor of 380, no significant change in the binding energy or kinetics of either substrate
R92A
-
mutation causes a decrease in the rate constant for the chemical step by a factor of 35000. The mutation causes no significant change in the binding energy or binding kinetics of MgATP2-. It does not cause a significant change in the binding energy of 6-hydroxymethyl-7,8-dihydropterin either but causes a decrease in the association rate constant for the binding of 6-hydroxymethyl-7,8-dihydropterin by a factor of 1.4 and a decrease in the dissociation rate constant by a factor of 10
V83Gdel84-89
-
the deletion mutation does not have significant effects on the dissociation constants or the rate constants for the binding of the first substrate MgATP2- or its analogues. The dissociation constant of 6-hydroxymethyl-7,8-dihydropterin for the mutant increases by a factor of about 100, which is due to a large increase in the dissociation rate constant. The deletion mutation causes a shift of the rate-limiting step in the reaction and a decrease in the rate constant for the chemical step by a factor of 110000. The crystal structures reveal that the deletion mutation does not affect protein folding, but the catalytic center of the mutant is not fully assembled even upon the formation of the ternary complex and is not properly sealed. Loop 3 is dispensable for the folding of the protein and the binding of the first substrate MgATP2-, but is required for the assembling and sealing of the active center. The loop plays an important role in the stabilization of the ternary complex and is critical for catalysis
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
because the enzyme is essential for microorganisms but is absent from human and animals, the enzyme is an excellent target for developing antimicrobial agent
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