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Information on EC 2.7.6.3 - 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase and Organism(s) Escherichia coli and UniProt Accession P26281
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2.7.6.3 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinaseIUBMB Comments
Binds 2 Mg2+ ions that are essential for activity . The enzyme participates in the biosynthetic pathways for folate (in bacteria, plants, fungi, and some archaeal species, including the haloarchaea) and methanopterin (in some archaeal species such as the Archaeoglobi and Methanobacteria). 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 , the enzyme from the plant Arabidopsis thaliana harbours the activity of EC 2.5.1.15, dihydropteroate synthase , while the enzyme from yeast Saccharomyces cerevisiae is trifunctional with both of the two above mentioned activities .
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Word Map
- 2.7.6.3
-
pyrophosphorylation
- pyrophosphate
- pterins
-
folic
- dihydrofolate
-
bisubstrate
-
sulfa
- dihydroneopterin
- ampcpp
- tetrahydrofolate
-
induced-fit
- drug development
- medicine
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
Synonyms
6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (hppk), 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, cythppk/dhps, fthppk-dhps, 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase, hydroxymethyldihydropterin pyrophosphokinase (hppk), hppk/dhps, hydroxymethyldihydropteridine pyrophosphokinase, 6-hydroxymethylpterin pyrophosphokinase, dihydropterin pyrophosphokinase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase
-
6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase
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HPPK
-
2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase
-
-
-
-
6-hydroxy-7,8-dihydropterin pyrophosphokinase
-
-
-
-
7,8-dihydro-6-hydroxymethylpterin pyrophosphokinase
-
-
-
-
7,8-dihydroxymethylpterin-pyrophosphokinase
-
-
-
-
ATP:2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine 6'-pyrophosphotransferase
-
-
-
-
H2-pteridine-CH2-OH pyrophosphokinase
-
-
-
-
H2-pteridine-CH2OH pyrophosphokinase
-
-
-
-
HPPK
-
-
-
-
hydroxymethyldihydropteridine pyrophosphokinase
-
-
-
-
pyrophosphokinase, 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + 6-hydroxymethyl-7,8-dihydropterin = AMP + 6-hydroxymethyl-7,8-dihydropterin diphosphate
ATP + 6-hydroxymethyl-7,8-dihydropterin = AMP + 6-hydroxymethyl-7,8-dihydropterin diphosphate
during its catalytic cycle, the enzyme must assume at least five liganded forms: the apoenzyme form (without either of the substrates), the binary substrate complex with MgATP, the ternary substrate complex (Michaelis complex) with MgATP and 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine, the ternary product complex with AMP and 6-hydroxymethyl-7,8-dihydropterin diphosphate (HPPP), and the binary product complex with HPPP
ATP + 6-hydroxymethyl-7,8-dihydropterin = AMP + 6-hydroxymethyl-7,8-dihydropterin diphosphate
in the enzyme mechanism, the assembly of the pterin-binding pocket depends on ATP-dependent conformational changes in the three active site loops
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
diphosphate transfer
-
-
-
-
PATHWAY SOURCE
PATHWAYS
KEGG
-
-, -, -, -, -, -
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, fungi, and some archaeal species, including the haloarchaea) and methanopterin (in some archaeal species such as the Archaeoglobi and Methanobacteria). 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
37278-23-2
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
6-hydroxymethyl-7,8-dihydropterin + MgATP2-
6-hydroxymethyl-7,8-dihydropterin diphosphate + MgAMP
biosynthesis of folate cofactors
-
-
?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + 2-amino-7,8-dihydro-4-hydroxy-6-(diphosphooxymethyl)pteridine
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 6-hydroxymethyl-7,8-dihydropteridine diphosphate
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 7,8-dihydro-6-(diphosphooxymethyl)pteridine
-
-
-
?
ATP + 6-hydroxymethyl-7,8-dihydropterin
AMP + H+ + (7,8-dihydropterin-6-yl)methyl diphosphate
-
-
-
?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + 2-amino-7,8-dihydro-4-hydroxy-6-(diphosphooxymethyl)pteridine
-
-
-
-
?
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 6-hydroxymethyl-7,8-dihydropteridine diphosphate
-
-
-
-
?
dATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
dAMP + 2-amino-7,8-dihydro-4-hydroxy-6-(diphosphooxymethyl)pteridine
-
-
-
-
?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
-
-
-
?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
-
-
-
-
?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + 2-amino-7,8-dihydro-4-hydroxy-6-(diphosphooxymethyl)pteridine
key step in biosynthesis of folic acid
-
-
?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + 2-amino-7,8-dihydro-4-hydroxy-6-(diphosphooxymethyl)pteridine
ATP binding is followed by binding of 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
-
-
?
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 6-hydroxymethyl-7,8-dihydropteridine diphosphate
-
-
-
?
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 6-hydroxymethyl-7,8-dihydropteridine diphosphate
enzyme binds ATP first, followed by 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
-
?
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 6-hydroxymethyl-7,8-dihydropteridine diphosphate
the product 6-hydroxymethyl-7,8-dihydropterin diphosphate is an intermediate in the pathway for folic acid biosynthesis
-
?
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 6-hydroxymethyl-7,8-dihydropteridine diphosphate
ordered bi-bi mechanism with ATP as the first substrate
-
-
?
additional information
?
-
binding affinity for GTP and GMP, 75fold weaker than for ATP
-
-
?
additional information
?
-
-
binding affinity for GTP and GMP, 75fold weaker than for ATP
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
6-hydroxymethyl-7,8-dihydropterin + MgATP2-
6-hydroxymethyl-7,8-dihydropterin diphosphate + MgAMP
biosynthesis of folate cofactors
-
-
?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + 2-amino-7,8-dihydro-4-hydroxy-6-(diphosphooxymethyl)pteridine
key step in biosynthesis of folic acid
-
-
?
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 6-hydroxymethyl-7,8-dihydropteridine diphosphate
the product 6-hydroxymethyl-7,8-dihydropterin diphosphate is an intermediate in the pathway for folic acid biosynthesis
-
?
ATP + 6-hydroxymethyl-7,8-dihydropteridine
AMP + 6-hydroxymethyl-7,8-dihydropteridine diphosphate
-
-
-
-
?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
-
-
-
?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
AMP + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mg2+
Mg2+
-
required for binding of nucleotides and for the binding of 6-hydroxymethyl-7,8-dihydropteridine
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2-(((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)methyl)-5-fluorobenzonitrile
-
2-(((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)methyl)-benzonitrile
-
2-((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)-N,N-diethylacetamide
-
2-((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)-N,N-dimethylacetamide
-
2-((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)-N-ethyl-N-methylpropanamide
-
2-((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)-N-isobutylacetamide
-
2-((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)-N-methylacetamide
-
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
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
2-amino-8-((2-(2-oxopyrrolidin-1-yl)ethyl)thio)-1,9-dihydro-6H-purin-6-one
-
2-amino-8-((2-(4-bromophenyl)-2-oxoethyl)thio)-1,9-dihydro-6H-purin-6-one
-
2-amino-8-((2-(trifluoromethyl)benzyl)thio)-1,9-dihydro-6Hpurin-6-one
-
2-amino-8-((2-fluoro-3-methylbenzyl)thio)-1,9-dihydro-6Hpurin-6-one
-
2-amino-8-((2-fluoro-4-methoxybenzyl)thio)-1,9-dihydro-6H-purin-6-one
-
2-amino-8-((2-fluoro-5-methylbenzyl)thio)-1,9-dihydro-6Hpurin-6-one
-
2-amino-8-((3,3,3-trifluoro-2-hydroxypropyl)thio)-1,9-dihydro-6H-purin-6-one
-
2-amino-8-((3,5-bis(trifluoromethyl)benzyl)thio)-1,9-dihydro-6H-purin-6-one
-
2-amino-8-((3-(trifluoromethoxy)benzyl)thio)-1,9-dihydro-6H-purin-6-one
-
2-amino-8-((4-(trifluoromethoxy)benzyl)thio)-1,9-dihydro-6H-purin-6-one
-
2-amino-8-((4-(trifluoromethyl)benzyl)thio)-1,9-dihydro-6Hpurin-6-one
-
2-amino-8-((4-fluoro-2-methylbenzyl)thio)-1,9-dihydro-6Hpurin-6-one
-
2-amino-8-((4-fluoro-3-methylbenzyl)thio)-1,9-dihydro-6Hpurin-6-one
-
2-amino-8-[2-(2-methylphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
2-amino-8-[2-(3-methylphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
2-amino-8-[2-(4-fluorophenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
2-amino-8-[2-(4-hydroxyphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
2-amino-8-[2-(4-methoxyphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
2-amino-8-[2-(4-methylphenyl)-2-oxoethoxy]-1,4,5,9-tetrahydro-6H-purin-6-one
-
3-(((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)methyl)-benzoic acid
-
3-(((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)methyl)-benzonitrile
-
4-(((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)methyl)-3-fluorobenzonitrile
-
4-(((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)methyl)-benzonitrile
-
4-[(2-amino-6-oxo-4,5,6,9-tetrahydro-1H-purin-8-yl)methyl]benzonitrile
-
4-[[(2-amino-6-oxo-4,5,6,9-tetrahydro-1H-purin-8-yl)oxy]acetyl]benzonitrile
-
5'-(1-[2-[(2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridine-6-carbonyl)amino]ethyl]-2-carboxypiperidine-4-sulfonyl)-5'-deoxyadenosine
bisubstrate inhibitor, Kd value is 0.000047 microM, compound is not sufficiently stable under the experimental conditions
5'-S-(1-[2-[(2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridine-6-carbonyl)amino]ethyl]-2-carboxypiperidin-4-yl)-5'-thioadenosine
bisubstrate inhibitor, the carboxylic group in the piperidine system mimics the gamma-phosphate of AMPCPP and the carboxylic group interacts with the side chains of H115, Y116, and R121
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
-
5-(((2-amino-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)methyl)-2-fluorobenzonitrile
-
8-((2-(1,3-dioxan-2-yl)ethyl)thio)-2-amino-1,9-dihydro-6Hpurin-6-one
-
8-((2-([1,1'-biphenyl]-4-yl)-2-oxoethyl)thio)-2-amino-1,9-dihydro-6H-purin-6-one
-
alpha,beta-methyleneadenosine triphosphate
competitive with respect to ATP
additional information
identification of several enzyme inhibitors that contain an aryl substituted 8-thioguanine scaffold. The compounds engage the enzyme pterin-binding pocket and an induced cryptic pocket, preliminary structure activity relationship profile, binding structures, overview
-
additional information
synthesis of a series of S-functionalized 8-mercaptoguanine analogues as substrate-site inhibitors of enzyme HPPK, binding structure analysis, and comparison of inhibition efficiency with the enzymes from Staphylococcus aureus and Escherichia coli, KD values, overview
-
additional information
-
synthesis of a series of S-functionalized 8-mercaptoguanine analogues as substrate-site inhibitors of enzyme HPPK, binding structure analysis, and comparison of inhibition efficiency with the enzymes from Staphylococcus aureus and Escherichia coli, KD values, overview
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
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
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
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
in 100 mM Tris, pH 8.3, at 23°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
physiological function
additional information
metabolism
6-hydroxymethyl-7,8-dihydropterin diphosphokinase catalyzes the first reaction in the folate biosynthetic pathway
metabolism
essential enzyme in the microbial folate biosynthetic pathway
physiological function
essential enzyme in the microbial folate biosynthetic pathway
additional information
movements of Arg82 and Arg92 adjacent to the phosphate groups of the bound ATP play an essential role in HPPK catalysis
additional information
putative large-scale induced-fit conformational change of enzyme HPPK. The loops 2 and 3 remain rather flexible when the enzyme is in its apoform, but loops 2 and 3 adopt the open, semi-open, and closed conformations throughout its catalytic cycle, flexibility of the three catalytic loops in the apoform of the enzyme. Molecular dynamics simulations of the apoenzyme at two different temperatures, overview
additional information
-
putative large-scale induced-fit conformational change of enzyme HPPK. The loops 2 and 3 remain rather flexible when the enzyme is in its apoform, but loops 2 and 3 adopt the open, semi-open, and closed conformations throughout its catalytic cycle, flexibility of the three catalytic loops in the apoform of the enzyme. Molecular dynamics simulations of the apoenzyme at two different temperatures, overview
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
the enzyme has an alphabetaalpha fold with a central six-stranded beta-sheet sandwiched by two alpha-helices on either side
additional information
-
the enzyme has an alphabetaalpha fold with a central six-stranded beta-sheet sandwiched by two alpha-helices on either side
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 19°C 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
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+
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 19°C
in complex with inhibitors (2R,4R)-1-(2-(2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridine-6-carboxamido)ethyl)-4-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)thio) piperidine-2-carboxylic acid and (2R,4R)-1-(2-(2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydropteridine-6-carboxamido)ethyl)-4-((((2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)sulfonyl) piperidine-2-carboxylic acid
molecular dynamics simulations to investigate the loop dynamics in the binary HPPK-MgATP complex. With loop 3 closed, multiple conformations of loop 2, including the open, semiopen, and closed forms, are all accessible to the binary complex. Loop 3 is unlikely to be opened due to the blockage caused by the binding of ATP
purified recombinant detagged enzyme in complex with different inhibitors and AMPCPP, sitting-drop vapor diffusion method, mixing of 2.2 mg/ml protein in 20 mM Tris-HCl buffer, pH 8.0, 100 mM NaCl, with reservoir solution containing 20% w/v PEG 4000, 0.1 M TrisCl, and 0.172 M CaCl2, or 0.1 M HEPES-NaOH, pH 7.5, 0.2 M CaCl2, and 25-30% PEG 4000, 16-20°C, addition of 2 mM MgCl2, 1 mM AMPCPP, and 1 mM of inhibitor, X-ray diffraction structure determination and analysis, molecular replacement
structure-based model upon ligand binding, molecular dynamics simulation. HPPK can switch to the activated holo state upon the ordered binding of ligands ATP and HP. The ligand-free HPPK can execute large-scale conformational fluctuations around the apo and open basins. ATP prefers to bind to the open conformations and promotes the population of the open state. Only when both ligands are bound, the conformational transitions among all of the three native states can emerge. Higher temperatures promote population shift, while the induced fit pathway is always the predominant activation route of the HPPK system
V83Gdel84-89 and its complex with alpha,beta--methyleneadenosine triphosphate and 6-hydroxymethyl-7,8-dihydropterin are crystallized at 19°C using the hanging-drop vapor-diffusion technique
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
R84A
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
W89A
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
additional information
the enzyme is minimally biotinylated by reaction with EZ-Link sulfo-NHS-LC-LC-biotin
R84A
little changes in the dissociation constants and kinetic constants of the HPPK-catalyzed reaction
R84A
the mutation causes little changes in either dissociation constants or kinetic constants of the enzyme-catalyzed reaction except that the rate constant for the chemical step of the forward reaction decreases by a factor of 4
W89A
mutation increases the Kd for the binding of MgATP2- by a factor of 3, whereas the Kd for 6-hydroxymethyl-7,8-dihydropterin increases by a factor of 6, which is due to the increase in the dissociation rate constant. The mutation decreases the rate constant for the chemical step of the forward reaction by a factor of 15 and the rate constant for the chemical step of the reverse reaction by a factor of 25. The crystal structures of W89A show that W89A has different conformations in loops 2 and 3, but the critical catalytic residues are positioned for catalysis
W89A
the mutation does not have any significant effects on the Kd and the rate constants for the binding of MgATP, but the Kd for 6-hydroxymethyl-7,8-dihydropteridine of the mutant increases by a factor of 6.5. The mutation decreases the rate constant for the chemical step of the forward reaction by a factor of about 23 and the rate constant for the chemical step of the reverse reaction by a factor of about 33
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by TEV protease, and gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
drug development
the enzyme is a target for antimicrobial development
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Richey, D.P.; Brown, G.M.
The biosynthesis of folic acid. IX. Purification and properties of the enzymes required for the formation of dihydropteroic acid
J. Biol. Chem.
244
1582-1592
1969
Escherichia coli
Talarico, T.L.; Dev, I.K.; Dallas, W.S.; Ferone, R.; Ray, P.H.
Purification and partial characterization of 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase and 7,8-dihydropteroate synthase from Escherichia coli MC4100
J. Bacteriol.
173
7029-7032
1991
Escherichia coli
Bock, L.; Bartels, R.
New method for the purification of 7,8-dihydro-6-hydroxymethylpterin pyrophosphokinase (E.C. 2.7.6.3) from Escherichia coli
J. Chromatogr.
26
206-209
1983
Escherichia coli
-
Bermingham, A.; Bottomley, J.R.; Primrose, W.U.; Derrick, J.P.
Equilibrium and kinetic studies of substrate binding to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli
J. Biol. Chem.
275
17962-17967
2000
Escherichia coli (P26281), Escherichia coli
Ballantine, S.P.; Volpe, F.; Delves, C.J.
The hydroxymethyldihydropterin pyrophosphokinase domain of the multifunctional folic acid synthesis Fas protein of Pneumocystis carinii expressed as an independent enzyme in Escherichia coli: refolding and characterization of the recombinant enzyme
Protein Expr. Purif.
5
371-378
1994
Escherichia coli, Pneumocystis carinii
Li, Y.; Gong, Y.; Shi, G.; Blaszczyk, J.; Ji, X.; Yan, H.
Chemical transformation is not rate-limiting in the reaction catalyzed by Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase
Biochemistry
41
8777-8783
2002
Escherichia coli
Li, Y.; Wu, Y.; Blaszczyk, J.; Ji, X.; Yan, H.
Catalytic roles of arginine residues 82 and 92 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: site-directed mutagenesis and biochemical studies
Biochemistry
42
1581-1588
2003
Escherichia coli
Garcon, A.; Bermingham, A.; Lian, L.Y.; Derrick, J.P.
Kinetic and structural characterization of a product complex of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli
Biochem. J.
380
867-873
2004
Escherichia coli (P26281), Escherichia coli
Blaszczyk, J.; Li, Y.; Shi, G.; Yan, H.; Ji, X.
Dynamic roles of arginine residues 82 and 92 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: crystallographic studies
Biochemistry
42
1573-1580
2003
Escherichia coli (P26281), Escherichia coli
Blaszczyk, J.; Li, Y.; Wu, Y.; Shi, G.; Ji, X.; Yan, H.
Essential roles of a dynamic loop in the catalysis of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase
Biochemistry
43
1469-1477
2004
Escherichia coli (P26281)
Li, Y.; Blaszczyk, J.; Wu, Y.; Shi, G.; Ji, X.; Yan, H.
Is the critical role of loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase in catalysis due to loop-3 residues arginine-84 and tryptophan-89? Site-directed mutagenesis, biochemical, and crystallographic studies
Biochemistry
44
8590-8599
2005
Escherichia coli (P26281), Escherichia coli
Yang, R.; Lee, M.C.; Yan, H.; Duan, Y.
Loop conformation and dynamics of the Escherichia coli HPPK apo-enzyme and its binary complex with MgATP
Biophys. J.
89
95-106
2005
Escherichia coli (P26281), Escherichia coli
Li, G.; Felczak, K.; Shi, G.; Yan, H.
Mechanism of the conformational transitions in 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as revealed by NMR spectroscopy
Biochemistry
45
12573-12581
2006
Escherichia coli (P26281)
Shi, G.; Shaw, G.; Li, Y.; Wu, Y.; Yan, H.; Ji, X.
Bisubstrate analog inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: new lead exhibits a distinct binding mode
Bioorg. Med. Chem.
20
4303-4309
2012
Escherichia coli (P26281)
Shi, G.; Shaw, G.; Liang, Y.H.; Subburaman, P.; Li, Y.; Wu, Y.; Yan, H.; Ji, X.
Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New design with improved properties
Bioorg. Med. Chem.
20
47-57
2012
Escherichia coli (P26281)
Yan, H.; Ji, X.
Role of protein conformational dynamics in the catalysis by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase
Protein Pept. Lett.
18
328-335
2011
Escherichia coli (P26281)
Gao, K.; He, H.; Yang, M.; Yan, H.
Molecular dynamics simulations of the Escherichia coli HPPK apo-enzyme reveal a network of conformational transitions
Biochemistry
54
6734-6742
2015
Escherichia coli (P26281), Escherichia coli
Yun, M.K.; Hoagland, D.; Kumar, G.; Waddell, M.B.; Rock, C.O.; Lee, R.E.; White, S.W.
The identification, analysis and structure-based development of novel inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase
Bioorg. Med. Chem.
22
2157-2165
2014
Escherichia coli (P26281)
Dennis, M.L.; Pitcher, N.P.; Lee, M.D.; DeBono, A.J.; Wang, Z.C.; Harjani, J.R.; Rahmani, R.; Cleary, B.; Peat, T.S.; Baell, J.B.; Swarbrick, J.D.
Structural basis for the selective binding of inhibitors to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Staphylococcus aureus and Escherichia coli
J. Med. Chem.
59
5248-5263
2016
Escherichia coli (P26281), Escherichia coli, Staphylococcus aureus
Gao, K.; Jia, Y.; Yang, M.
A network of conformational transitions revealed by molecular dynamics simulations of the binary complex of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase with MgATP
Biochemistry
55
6931-6939
2016
Escherichia coli (P26281), Escherichia coli
Shi, G.; Shaw, G.X.; Zhu, F.; Tarasov, S.G.; Ji, X.
Bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase Transition state analogs for high affinity binding
Bioorg. Med. Chem.
29
115847
2020
Escherichia coli (P26281)
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