Information on EC 2.5.1.15 - dihydropteroate synthase

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

EC NUMBER
COMMENTARY hide
2.5.1.15
-
RECOMMENDED NAME
GeneOntology No.
dihydropteroate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(7,8-dihydropterin-6-yl)methyl diphosphate + 4-aminobenzoate = diphosphate + 7,8-dihydropteroate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
aryl group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Folate biosynthesis
-
-
Metabolic pathways
-
-
tetrahydrofolate biosynthesis
-
-
tetrahydrofolate metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
(7,8-dihydropterin-6-yl)methyl diphosphate:4-aminobenzoate 2-amino-4-hydroxy-7,8-dihydropteridine-6-methenyltransferase
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 plant Arabidopsis thaliana also harbors the activity of EC 2.7.6.3, 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase [4], while the enzyme from yeast Saccharomyces cerevisiae is trifunctional with the two above mentioned activities as well as EC 4.1.2.25, dihydroneopterin aldolase [3].
CAS REGISTRY NUMBER
COMMENTARY hide
9055-61-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
-
Q1BXC8
UniProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
strain BCG
A1KPU3
UniProt
Manually annotated by BRENDA team
no activity in Homo sapiens
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
-
prevalence and frequency of the dihydropteroate synthetase mutations associated with sulfadoxine-pyrimethamine resistance in southern Mozambique are examined between 1999 and 2004. The dihydropteroate synthetase double mutation frequency peaks in 2001 but declines to baseline levels by 2004. Parasites with both dihydrofolate reductase triple and dihydropteroate synthetase double mutations increase in 2001 but decrease by 2004. The peaking of sulfadoxine-pyrimethamine resistance markers in 2001 coincides with a sulfadoxine-pyrimethamine-resistant malaria epidemic in neighboring KwaZulu-Natal, South Africa. The decline in dihydropteroate synthetase (but not dihydrofolate reductase) mutations correspond with replacement of sulfadoxine-pyrimethamine with artemether-lumefantrine as malaria treatment policy in KwaZulu-Natal
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
(2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate + 4-aminobenzoate
diphosphate + 7,8-dihydropteroate
show the reaction diagram
2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine + 4-aminobenzoate
7,8-dihydropteroate + diphosphate
show the reaction diagram
2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + 4-aminobenzoate
diphosphate + 7,8-dihydropteroate
show the reaction diagram
2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + 4-aminobenzoylglutamate
diphosphate + dihydrofolate
show the reaction diagram
-
-
-
?
2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + sulfamethoxazole
diphosphate + dihydropterin-sulfamethoxazole
show the reaction diagram
-
-
-
?
2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + sulfanilamide
diphosphate + dihydropterin-sulfanilamide
show the reaction diagram
-
-
-
?
2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + sulfathiazole
diphosphate + dihydropterin-sulfathiazole
show the reaction diagram
-
-
-
?
4-aminobenzoate + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
diphosphate + 7,8-dihydropteroate
show the reaction diagram
-
-
-
-
?
6-hydroxymethyl-7,8-dihydropterin diphosphate + 4-aminobenzoate
diphosphate + 7,8-dihydropteroate
show the reaction diagram
-
pH 7.5, folate biosynthesis
coupled with diphosphate-dependent phosphofructokinase, aldolase, triosephosphate isomerase, alpha-glycerophosphate dehydrogenase, in presence of NADH and D-fructose 6-phosphate, decrease in NADH monitored as change of absorbance at 340 nm
-
?
dapson + Mg-6-hydroxymethyl-7,8-dihydropterin diphosphate
diphosphate + 2-amino-6-[([4-[(4-aminophenyl)sulfonyl]phenyl]amino)methyl]-7,8-dihydropteridin-4(3H)-one
show the reaction diagram
-
-
-
-
?
p-aminobenzoate + 6-hydroxymethyl-7,8-dihydropterin-diphosphate
diphosphate + 7,8-dihydropteroate
show the reaction diagram
p-aminobenzoic acid + 6-hydroxymethyl-7,8-dihydropterin diphosphate
diphosphate + 7,8-dihydropteroate
show the reaction diagram
substrate binding order: 6-hydroxymethyl-7,8-dihydropterin diphosphate binds prior to p-aminobenzoic acid, 6-hydroxymethyl-7,8-dihydropterin diphosphate binding with KD: 33 +/-6 microM (k(on): 260000 1/M*s, k(off): 8.7 1/s) as revealed by fluorescence spectroscopy, p-aminobenzoic acid binding to diphosphate-enzyme complex: KD: 0.13 +/-0.02 microM
diphosphate-binding allows binding of p-aminobenzoic acid or p-aminobenzoic acid analogues, diphosphate binding with KD: 350 +/-20 microM (k(on): 56000 1/M*s, k(off): 21 1/s) as revealed by fluorescence spectroscopy
-
?
sulfadiazine + 6-hydroxymethyl-7,8-dihydropterin diphosphate
diphosphate + 4-[[(2-amino-4-oxo-3,4,7,8-tetrahydropteridin-6-yl)methyl]amino]-N-pyrimidin-2-ylbenzenesulfonamide
show the reaction diagram
-
-
-
-
?
sulfamethazole + 6-hydroxymethyl-7,8-dihydropterin diphosphate
diphosphate + 4-[[(2-amino-4-oxo-3,4,7,8-tetrahydropteridin-6-yl)methyl]amino]-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide
show the reaction diagram
-
-
-
-
?
sulfamethizole + 6-hydroxymethyl-7,8-dihydropterin diphosphate
?
show the reaction diagram
-
-
-
-
?
sulfamethoxazole + 6-hydroxymethyl-7,8-dihydropterin diphosphate
diphosphate + 4-[[(2-amino-4-oxo-3,4,7,8-tetrahydropteridin-6-yl)methyl]amino]-N-(5-methylisoxazol-3-yl)benzenesulfonamide
show the reaction diagram
-
-
-
-
?
sulfanilamide + 6-hydroxymethyl-7,8-dihydropterin diphosphate
diphosphate + 4-[[(2-amino-4-oxo-3,4,7,8-tetrahydropteridin-6-yl)methyl]amino]benzenesulfonamide
show the reaction diagram
-
-
-
-
?
sulfathiazole + 6-hydroxymethyl-7,8-dihydropterin diphosphate
diphosphate + 4-[[(2-amino-4-oxo-3,4,7,8-tetrahydropteridin-6-yl)methyl]amino]-N-1,3-thiazol-2-ylbenzenesulfonamide
show the reaction diagram
-
-
-
-
?
sulfisoxazole + 6-hydroxymethyl-7,8-dihydropterin diphosphate
?
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
(2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate + 4-aminobenzoate
diphosphate + 7,8-dihydropteroate
show the reaction diagram
2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + 4-aminobenzoate
diphosphate + 7,8-dihydropteroate
show the reaction diagram
2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + 4-aminobenzoylglutamate
diphosphate + dihydrofolate
show the reaction diagram
-
-
-
?
2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + sulfamethoxazole
diphosphate + dihydropterin-sulfamethoxazole
show the reaction diagram
-
-
-
?
4-aminobenzoate + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
diphosphate + 7,8-dihydropteroate
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
amino acid V585 may be responsible for resistance of Plasmodium vivax to sulfadoxine
-
-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
flavin adenine dinucleotide
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KCl
-
0.4 M activates
NaCl
-
0.2 M activates
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(7-amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazin-3-yl)acetic acid
-
-
(7-amino-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazin-3-yl)acetic acid
-
-
2-(7-amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazin-3-yl)propanoate
-
binding structure, interactions with the DHPS module and the HPPK module, modeling, ovverview
2-(7-amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazin-3-yl)propanoic acid
-
-
2-(7-amino-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazin-3-yl)propanoic acid
-
-
2-amino-4-hydroxy-6-hydroxymethyl-dihydropteridine
-
-
3-(7-amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazin-3-yl)butanoic acid
-
-
3-(7-amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazin-3-yl)propanoic acid
-
-
3-(7-amino-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazin-3-yl)propanoic acid
-
-
4,4-Diaminodiphenyl sulfone
-
-
5-nitro-6-methylamino-isocytosine
-
-
-
6-hydroxymethylpterin monophosphate
-
6HMP, competitive inhibitor, 6-hydroxymethyl-7,8-dihydropterin diphosphate analogue
7,8-Dihydropteroic acid
7-amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazine-3-carboxylic acid
-
-
7-amino-3-(1-carboxyethyl)-1-methyl-pyrimido (4,5-c)-pyridazine-4,5(1H,6H)-dione
dapson
-
KD: 0.2 microM according to molecular modelling
dapsone
dihydrofolate
-
-
dihydrofolate monoglutamate
-
-
Guanidine HCl
-
0.25 M, 50% inhibition
KCl
-
above 0.4 M
NaCl
-
above 0.2 M
p-Aminobenzoate
-
-
p-Aminobenzoylglutamate
-
weak
p-Aminosalicylate
p-Aminosalicylic acid
phosphanilic acid
potassium 4-([(2-amino-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-6-yl)-(2-phosphonoethyl)-amino]-methyl)-benzoate
-
the oxidized analogue shows significant DHPS inhibition and significant antimicrobial activity
potassium 4-([(2-amino-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-6-yl)-(3-phosphonopropyl)-amino]-methyl)-benzoate
-
the oxidized analogue shows significant DHPS inhibition and significant antimicrobial activity
Pteroic acid
-
PTA, product analog, binds pterin- and 4-aminobenzoate-binding regions
pyrimethamine
sulfacetamide
sulfachloropyridazine
sulfachlorpyridazine
-
-
Sulfadiazine
sulfadimethoxine
sulfadoxine
sulfadoxine-pyrimethamine
-
-
Sulfaguanidine
-
-
sulfamerazine
sulfamethoxazole
sulfamethoxypyridazine
sulfamoxisole
-
effective against wild-type enzyme and mutant enzymes A437G/K540E, A437G, S436F/A437G/A613S and S436F/A437G/A613T. Ineffective against A437G/A581G
-
sulfamoxole
sulfanilamide
Sulfanilic acid
-
-
sulfapyridine
sulfaquinoxaline
sulfaquinoxazoline
-
-
-
sulfathiazole
sulfisoxazole
sulfonamide
Sulfonamides
-
act as competitive inhibitors with respect to the 4-amino benzoic acid substrate within the DHPS enzyme active site. The 4-amino benzoic acid/sulfonamide binding site is formed close to the protein surface by flexible protein loops facilitating rapid development of sulfonamide resistance. Study of inhibitors designed to target the conserved central pterin binding site within DHPS, molecular dynamics simulation and molecular modeling, overview. Design and synthesis of transition state analogues with ability to mimic the intermediate transient carbocation by the incorporation of a basic amine at the 6-position of the pterin ring
sulfpyridine
-
effective against wild-type enzyme. Ineffective against mutants A437G, A437G/A581G, A437G/K540E, S436F/A437G/A613S and S436F/A437G/A613T
-
tetrahydrofolate monoglutamate
-
-
trimethoprim-sulfamethoxazole
-
-
Trp-Lys
-
WK, highly potent with KD: 0.23 nM according to structure-based molecular modelling and docking, overlaps with pterin monophosphate and pteroic acid binding regions, highly selective for microbial DHPS compared to human pterin and folate-binding enzymes (dihydrofolate reductase, thymidylate synthase)
Urea
-
0.9 M, 50% inhibition
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Sulfadiazine
-
reaction rate increased by sulfadiazine sodium salt, sulfamethoxazole, sulfamethazole, sulfanilamide, sulfathiazole sodium salt in presence of both substrates
sulfamethoxazole
-
reaction rate increased by sulfadiazine sodium salt, sulfamethoxazole, sulfamethazole, sulfanilamide, sulfathiazole sodium salt in presence of both substrates
sulfanilamide
-
reaction rate increased by sulfadiazine sodium salt, sulfamethoxazole, sulfamethazole, sulfanilamide, sulfathiazole sodium salt in presence of both substrates
sulfathiazole
-
reaction rate increased by sulfadiazine sodium salt, sulfamethoxazole, sulfamethazole, sulfanilamide, sulfathiazole sodium salt in presence of both substrates
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0036 - 0.0514
(2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
0.0004 - 0.00122
2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
0.0014 - 0.0019
2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate
0.0005 - 0.0038
4-Aminobenzoate
0.0025 - 0.0028
4-Aminobenzoic acid
0.0027
6-hydroxymethyl-7,8-dihydropterin diphosphate
-
100 microM 4-aminobenzoate, 25 C
0.03
6-hydroxymethyldihydropteridine diphosphate
-
-
0.000058
dapson
-
in absence of 4-aminobenzoate
0.0026 - 0.008
p-Aminobenzoate
0.00037 - 4.02
p-Aminobenzoic acid
0.00289
Sulfadiazine
-
in absence of 4-aminobenzoate
0.0007
sulfamethizole
-
in absence of 4-aminobenzoate
0.00012 - 0.0027
sulfamethoxazole
0.0006 - 0.0048
sulfanilamide
0.00033 - 0.001
sulfathiazole
0.0015
sulfisoxazole
-
in absence of 4-aminobenzoate
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.032 - 0.069
(2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
0.109
4-Aminobenzoate
Bacillus anthracis
-
100 microM DHP-PP, 25 C
0.0578
dapson
Bacillus anthracis
-
in absence of 4-aminobenzoate
0.1243
Sulfadiazine
Bacillus anthracis
-
in absence of 4-aminobenzoate
0.079
sulfamethizole
Bacillus anthracis
-
in absence of 4-aminobenzoate
0.12
sulfamethoxazole
Bacillus anthracis
-
in absence of 4-aminobenzoate
0.08
sulfanilamide
Bacillus anthracis
-
in absence of 4-aminobenzoate
0.0887
sulfathiazole
Bacillus anthracis
-
in absence of 4-aminobenzoate
0.11
sulfisoxazole
Bacillus anthracis
-
in absence of 4-aminobenzoate
additional information
additional information
Bacillus anthracis
-
linear dependency on DHPS concentration when concentration of enzyme versus initial velocity in the coupled enzymatic assay
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.08 - 6.23
(2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.043
2-amino-4-hydroxy-6-hydroxymethyl-dihydropteridine
-
-
0.0036
6-hydroxymethylpterin monophosphate
-
100 microM 4-aminobenzoate
0.005 - 0.011
7,8-dihydropteroate
0.000011 - 1.29
dapsone
0.015
folate
-
-
1
p-Aminobenzoylglutamate
-
-
0.0082 - 0.023
p-Aminosalicylic acid
0.0941
sulfacetamide
-
-
0.0015 - 0.94
sulfachloropyridazine
0.0068
Sulfadiazine
-
-
0.0018
sulfadimethoxine
-
-
0.011
sulfadoxine
-
-
0.069
Sulfaguanidine
-
-
0.0048
sulfamerazine
-
-
0.000028 - 1.17
sulfamethoxazole
0.000025 - 0.0033
sulfamethoxypyridazine
0.0027
sulfamoxole
-
-
0.00085 - 0.13
sulfanilamide
0.14
Sulfanilic acid
-
-
0.0019
sulfapyridine
-
-
0.0016
sulfaquinoxaline
-
-
0.025
sulfaquinoxazoline
-
-
-
0.0006 - 0.5
sulfathiazole
0.0076
sulfisoxazole
-
-
0.00053
sulfonamide
-
-
additional information
additional information
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0000483
-
crude extract of Escherichia coli MG1655
0.005
+/-3, assumed inactive because 100times less than folP1 gene product
0.174
-
recombinant enzyme
0.425
-
-
0.596
-
recombinant enzyme
1.25
-
recombinant bifunctional fusion protein encoding dihydropterin pyrophosphokinase and dihyropteroate synthase domains of the trifunctional enzyme dihydroneopterin aldolase-dihydropterin pyrophosphokinase-dihydropteroate synthase
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
-
dihydropteroate synthase activity of the recombinant bifunctional fusion protein consisting of dihydropterin diphosphokinase and dihyropteroate synthase domains
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 10.5
-
70% of maximal activity at pH 7.0 and 10.5
7 - 9.3
-
pH 7.0: about 50% of maximal activity, pH 9.3: about 60% of maximal activity, dihydropteroate synthase activity of the recombinant bifunctional fusion protein encoding dihydropterin pyrophosphokinase and dihyropteroate synthase domains of the trifunctional enzyme dihydroneopterin aldolase-dihydropterin pyrophosphokinase-dihydropteroate synthase
8.5 - 10.5
-
pH 8.5: about 40% of maximal activity, pH 10.5: about 85% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 55
-
30C: about 40% of maximal activity, 55C: about 10% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.11
-
recombinant hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
A1KPU3
logarithmic growing bacteria, Western blotting; qiescent bacteria, lower expression level under anaerobic conditions compared to nutrient starvation, Western blotting
Manually annotated by BRENDA team
additional information
-
bronchoalveolar lavage isolates from AIDS patient in Brazil
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus anthracis (strain A0248)
Burkholderia cenocepacia (strain ATCC BAA-245 / DSM 16553 / LMG 16656 / NCTC 13227 / J2315 / CF5610)
Burkholderia cenocepacia (strain ATCC BAA-245 / DSM 16553 / LMG 16656 / NCTC 13227 / J2315 / CF5610)
Coxiella burnetii (strain RSA 493 / Nine Mile phase I)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
Streptococcus pneumoniae (strain ATCC BAA-255 / R6)
Streptococcus pneumoniae (strain ATCC BAA-255 / R6)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
29000
recombinant, hexa-His tagged enzyme, SDS-PAGE
33000
recombinant, hexa-His tagged enzyme, SDS-PAGE
34070
deduced from primary structure
50000
-
gel filtration
52000 - 54000
gel filtration
52000
-
polyacrylamide gel electrophoresis
68000
Q1BXC8
recombinant enzyme, gel filtration
71500
-
polyacrylamide gel electrophoresis
75000
recombinant hexa-His tagged enzyme, gel filtration chromatography
83000
-
bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase, gel filtration
110000
-
recombinant bifunctional fusion protein encoding dihydropterin pyrophosphokinase and dihyropteroate synthase domains of the trifunctional enzyme dihydroneopterin aldolase-dihydropterin pyrophosphokinase-dihydropteroate synthase, gel filtration
190000
-
Sephadex chromatography
222000
-
gel filtration
280000 - 300000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
monomer
-
1 * 50509, sequence calculation
additional information
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primary and secondary structures of the HPPK-DHPS bifunctional enzyme, structure comparisons, three-dimensional structure, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant apo-enzyme and enzyme in complex with inhibitor sulphadoxine or product 7,8-dihydropteroate, sitting drop vapor diffusion method, mixing of 0.001 ml of protein solution, containing 7.5 mg/ml protein in 50 mM Tris-HCl, 250 mM NaCl pH 7.5, with 0.001 ml of reservoir solution, containing 0.1 M Tris-HCl, pH 8, 10% PEG 8000, 0.3 M MgCl2, 4C, X-ray diffraction structure determinationation and analysis at 1.95-2.35 A resolution, molecular replacement and modeling
Q1BXC8
apo-enzyme, and enzyme in complex with HPPK substrate 6-hydroxymethyl-7,8-dihydropterin or inhibitor 2-(7-amino-1-methyl-4,5-dioxo-1,4,5,6-tetrahydorpyrimido[4,5-c]pyridazin-3-yl)propanoic acid, X-ray diffraction structure determination and analysis at 2.2-2.3 A resolution
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PDB: 2VP8, conforms classical triosephosphate isomerase (TIM) barrel arrangement, 6-hydroxymethyl-7,8-dihydropterin-diphosphate-binding pocket occupied by the histidine 220 side chain compared to leucine 180 in the functional ortholog MtDHPS (PDB: 1EYE), crystals: space group P3(1)21, 2 molecules per asymmetric unit, noncrystallographic dimer in the crystal structure, unit cell parameters: a, b: 80.73, c: 215.94, hanging drop vapour method: 25% ethylene glycole + protein solution (16 mg/ml), 2 days, 18C
structure-based molecular modelling of the missing loop 2 (PDB: 1EYE) using Escherichia coli DHPS (PDB: 1AJZ) for further docking approaches, (i) extraction and docking of pterin monophosphate (PtP), KD(PtP): 0.5 microM, (ii) ternary complex of dapson docked with enzyme-PtP complex, KD(dapson): 0.2 microM, contacts with residues S53, R54, P55, F182, K213, (iii) ternary complex of pteroic acid (PTA) docked with enzyme-PtP complex, contacts with pterin- and 4-aminobenzoate-binding regions, (iv) inhibitor complex with dipeptide Trp-Lys (WK), contacts with residues E51, S53, D21, D86, K213, R253, and four water molecules, KD(WK): 0.23 nM
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more putative structural model of five mutations in DHPS to explain sulfadoxine resistance, structural model based on crystal structures of Saccharomyces cerevisiae (PPPK-DHPS), Mycobacterium tuberculosis (DHPS), Bacillus anthracis (DHPS), and Escherichia coli (PPPK)
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a complex of the purified bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/dihydropteroate synthase with a pterin monophosphate substrate analogue, structure solved by molecular replacement and refined to 2.3 A resolution
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recombinant bifunctional fusion protein encoding dihydropterin pyrophosphokinase and dihyropteroate synthase domains of the trifunctional enzyme dihydroneopterin aldolase-dihydropterin pyrophosphokinase-dihydropteroate synthase
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apoenzyme (PDB: 2VEF), complex with 6-hydroxymethyl-7,8-dihydropterin monophosphate (DHPP) (PDB: 2VEG), TIM alpha/beta barrel fold with highly conserved 6-hydroxymethyl-7,8-dihydropterin diphosphate-binding pocket, crystals: space group P2(1)2(1)2(1), unit cell parameters: a: 45, b: 90, c: 137, loop 1 and 2 highly flexible, dimer of two identical monomers in the asymmetric unit, in complex with DHPP only one monomer of the dimer has substrate bound wide-scale rearrangement of active site upon 6-hydroxymethyl-7,8-dihydropterin diphosphate (DHPPP) binding mediated by diphosphate moiety, hanging-drop method: 2 microlitre protein solution (13 mg/ml) + 2 microl precipitant (0.2 M ammonium iodide, 20% (w/v) poly(ethylene glycol) 3350) +/-2.5 mM DHPPP (hydrolysis to DHPP during crystallization), 7-14 days, molecular replacement-based structure determination
apo-structure and structure of the complex with pteroate, analysis, overview
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 10
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stable, dihydropteroate synthase activity of the recombinant bifunctional fusion protein encoding dihydropterin pyrophosphokinase and dihyropteroate synthase domains of the trifunctional enzyme dihydroneopterin aldolase-dihydropterin pyrophosphokinase-dihydropteroate synthase
663313
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
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inactivation above
60
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10 min, loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C and -80C stable for 3 months in 20 mM TrisHCl, pH 8.0 buffer containing 10% or 20% glycerol
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-20C or -70C, 20 mM Tris/HCl, pH 8.0, 5 weeks
-20C or -80C, in buffer without glycerol, 30% and 50% of the enzyme activity is lost when stored for 6 months
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-20C, enzyme remains active in 50% glycerol, 1 mM MgCl2, 5 mM 2-mercaptoethanol, stable for long term-storage, dihydropteroate synthase activity of the recombinant bifunctional fusion protein encoding dihydropterin pyrophosphokinase and dihyropteroate synthase domains of the trifunctional enzyme dihydroneopterin aldolase-dihydropterin pyrophosphokinase-dihydropteroate synthase
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-20C, sucrose, indefinitely stable
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-80C, pH 7.0, 20% glycerol, 10-30% loss of activity in 1 month
4C, or -20C, without glycerol, enzyme denaturates, dihydropteroate synthase activity of the recombinant bifunctional fusion protein encoding dihydropterin pyrophosphokinase and dihyropteroate synthase domains of the trifunctional enzyme dihydroneopterin aldolase-dihydropterin pyrophosphokinase-dihydropteroate synthase
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
anion exchange and affinity column
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enzyme from recombinant clones in Sephacryl S-300 HR, enzyme isolated form Escherichia coli, several chromatographic columns
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gel filtration and ion-exchange chromatography
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gel filtration on Sephadex G-100
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His-tagged recombinant protein by fast-performance liquid chromatography
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mycobacterial enzymes, from DEAE-Sepharose and other chromatography steps
overexpression of the N-terminal His6 fusion protein in Escherichia coli, coexpression with chaperone genes to increase solubility
precipitation from bacterial lysate by ammonium sufate (50% saturation), resuspension in Tris/HCl pH 8, Resource Q ion-exchange chromatography (elution with NaCl gradient), dialysis, Mono Q ion-exchange chromatography (elution with NaCl gradient), for crystallisation followed by Superdex 200 gel-filtration chromatography
precipitation with ammonium sulfate followed by column chromatography
Q-Sepharose column
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recombinant bifunctional fusion protein encoding dihydropterin pyrophosphokinase and dihyropteroate synthase domains of the trifunctional enzyme dihydroneopterin aldolase-dihydropterin pyrophosphokinase-dihydropteroate synthase
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recombinant enzyme, affinity column
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)by nickel affinity chromatography and gel filtration
Q1BXC8
recombinant hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase
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recombinant, hexa-His tagged enzyme from bacterial lysate by nickel-nitrilotriacetic acid agarose, elution with 200 mM imidazole, stored at -80C in 10% glycerol or further purified on by Superdex 200 16/60 gel filtration chromatography; recombinant, hexa-His tagged enzyme from bacterial lysate by nickel-nitrilotriacetic acid agarose, elution with 200 mM imidazole, stored at -80C in 10% glycerol or further purified on by Superdex 200 GL 10/300 gel filtration chromatography
Superdex-200 HR and other chromatographic columns
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a single polypeptide, Fas protein, expressed in cultured Spodoptera frugiperda SF9 insect cells
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cDNA encoding dihydropterin pyrophosphokinase and dihyropteroate synthase domains of the trifunctional enzyme dihydroneopterin aldolase-dihydropterin pyrophosphokinase-dihydropteroate synthase is cloned. This bi-functional enzyme is expressed as a His6 fusion protein in Escherichia coli
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cloning of a sulfonamide resistant strain
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crystal structure of the liganded enzyme and of the enzyme in complex with 6-hydroxymethyl-pterine-diphosphate, 6-hydroxymethyl-pterine-monophosphate, 6-methylamino-5-nitroisocytosine, or pteroic acid. Hanging drop vapor diffusion method, the crystals are space group P6(2)22 with cell dimensions a = b = 97.4 A, c = 263.6 A
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engineering dihydropteroate synthase for efficient expression on M13 phage
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expressed in Escherichia coli
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expressed under the control of the T5 promoter in a DHPS-deficient Escherichia coli strain
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expression in different Escherichia coli strains
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expression in dihydroxypteroate synthase deficient strain of Escherichia coli
expression in Escherichia coli
expression in Escherichia coli as N-terminal His6 fusion; expression in Saccharomyces cerevisiae Fol1 knockout Y26466
expression in Escherichia coli C600 and in Escherichia coli folP knockout mutant; expression in Escherichia coli C600 and in Escherichia coli folP knockout mutant
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expression of recombinant enzyme in Escherichia coli
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from genomic DNA (strain H37Rv) in pET-21a for expression with C-terminal hexa-His tag in Escherichia coli BL21 (DE3); from genomic DNA (strain H37Rv) in pQE60 for expression with C-terminal hexa-His tag in Escherichia coli M15
from Streptococcus pneumoniae for expression in Escherichia coli XL2-Blue
gene encoding bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/dihydropteroate synthase, cloned and expressed in Escherichia coli
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recombinant expression of the His-tagged enzyme in Escherichia coli strain BL21(DE3)
Q1BXC8
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D96N
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mutation improves phage display efficiency of the enzyme
D96N/C137I/C172M/C242A
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130fold improvement in phage display efficiency compared to wild-type enzyme
G58A
site-directed mutagenesis, cannot replace DHPR in Escherichia coli, affects the binding affinity for FMN
K31A
site-directed mutagenesis, can replace DHPR in Escherichia coli, affects the binding affinity for FMN
K51A
site-directed mutagenesis, can replace DHPR in Escherichia coli, affects the binding affinity for FMN
K92E
site-directed mutagenesis, can replace DHPR in Escherichia coli, affects the binding affinity for FMN
M28E
site-directed mutagenesis, can replace DHPR in Escherichia coli, affects the binding affinity for FMN
P55A
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mutation associated with dapsone resistance
P55L
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mutation associated with dapsone resistance
T53A
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mutation associated with dapsone resistance
T53I
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mutation associated with dapsone resistance
T53V
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mutation associated with dapsone resistance
A437G/A581G
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mutant enzyme is resistant against inhibition by sulfadoxine, sulfpyridine, sulfadimethoxine, sulfamethoxazole, sulfaquinoxaline and sulfisoxazole (inhibitors of wild-type enzyme)
A437G/K540E
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sulfadoxine, sulfpyridine, sulfadimethoxine, sulfamethoxazole, sulfaquinoxaline, sulfisoxazole, sulfanilamide, sulfamerazine, sulfathiazole, dapsone, sulfamoxisole, sulfachloropyridazine and sulfacetamide (inhibitors of wild-type enzyme)
A613S
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little association with between mutantion and sulfa drug resistance in patients
DELTA247-306
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deletion of the parasite-specific insertion: Km values for (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate and p-aminobenzoate are increased compared to mutant DELTA257-306 but still decreased compared to wild type, catalytic efficacy (kcat) is comparable to wild type
DELTA257-306
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deletion of the parasite-specific insertion: Km values for (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate and p-aminobenzoate are significantly decreased compared to wild type, catalytic efficacy (kcat) is at least 2fold decreased compared to wild type
DELTA74-80
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deletion of the parasite-specific insertion: Km values for (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate and p-aminobenzoate are 2 to 3 fold higher compared to wild type, catalytic efficacy (kcat) is at least 2fold decreased compared to wild type
DELTA74-86
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no DHPS activity is detected
K540E
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correlation of polymorphisms in Plasmodium falciparum dihydropteroate synthase and in vitro parasite susceptibility to sulfadoxine and pyrimethamine and in vivo treatment are analyzed: Patients with the dihydropteroate synthase K540E mutation are 2.6times as likely to fail treatment compared to patients having wild-type form form
K540N
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parasites collected during October 2005 have mutations associated with a lower level of pyrimethamine resistance and a higher level of sulfadoxine resistance, as well as a novel K540N mutation in PfDHPS gene. The emergence of this parasite population coincides with the widespread use of an additional antifolate drug, trimethoprim-sulfamethoxazole, to treat other infections during January-March 2005
S436A
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little association with between mutantion and sulfa drug resistance in patients
S436F/A437G/A613S
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sulfadoxine, sulfpyridine, sulfadimethoxine, sulfamethoxazole, sulfaquinoxaline, sulfisoxazole, sulfanilamide, sulfamerazine, sulfathiazole and dapsone (inhibitors of wild-type enzyme)
S436F/A437G/A613T
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sulfadoxine, sulfpyridine, sulfadimethoxine, sulfamethoxazole, sulfaquinoxaline, sulfisoxazole, sulfanilamide, sulfamerazine, sulfathiazole and dapsone (inhibitors of wild-type enzyme)
P519S
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mutant with implicated sulfa drug resistance
P57S
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the prevalence of DHPS mutations in Pneumocystis jirovecii strains isolated from South African Pneumocystis jirovecii pneumonia patients are examined. Mutations resulting in amino-acid substitutions Thr55Ala and/or Pro57Ser are detected in Pneumocystis jirovecii from 85/151 (56%) patients. The high frequency of PCP episodes with Pneumocystis jirovecii harbouring DHPS mutations in South Africa indicates that populations of this fungus are evolving under considerable selective pressure exerted by sulfa-containing antibiotics
T517A
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mutant with implicated sulfa drug resistance
T55A
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the prevalence of DHPS mutations in Pneumocystis jirovecii strains isolated from South African Pneumocystis jirovecii pneumonia patients are examined. Mutations resulting in amino-acid substitutions Thr55Ala and/or Pro57Ser are detected in Pneumocystis jirovecii from 85/151 (56%) patients. The high frequency of PCP episodes with Pneumocystis jirovecii harbouring DHPS mutations in South Africa indicates that populations of this fungus are evolving under considerable selective pressure exerted by sulfa-containing antibiotics
P559S
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mutant with implicated sulfa drug resistance
T557A
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mutant with implicated sulfa drug resistance
T557A/P559S
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mutant with implicated sulfa drug resistance
T557V/P559S
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mutant with implicated sulfa drug resistance
T597V/P599S
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the mutant has very low p-aminobenzoic acid dependence, short generation time and and high sulfamethoxazole resistance. Upregulated p-aminobenzoic acid synthesis is implicated as a mechanism for sulfa drug resistance
GS60
insertion of glycine-serine dipeptide into loop 2 beginning at position 60, sulfonamide resistant, no effect on diphosphate affinity, no effect on 6-hydroxymethyl-7,8-dihydropterin diphosphate binding: KD: 46 +/-5 microM (k(on): 260000 1/M*s, k(off): 12 1/s), reduced binding of p-aminobenzoic acid: KD: 16 +/-6 microM, no detectable binding of sulfamethoxazole
InsY63
insertion of tyrosine residue in loop 2, sulfonamide resistant, no effect on diphosphate affinity, no effect on 6-hydroxymethyl-7,8-dihydropterin diphosphate binding: KD: 48 +/-5 microM (k(on) = 240000 1/M*s, k(off): 11 1/sec), reduced binding of p-aminobenzoic acid: KD: 50 +/-6 microM, no detectable binding of sulfamethoxazole
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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the Bacillus anthracis DHPS pterin-binding pocket is analysed using five docking programs (FlexX, Surflex, Glide, GOLD, and DOCK) and nine scoring functions using pose selection/scoring and enrichment studies. Pose selection and scoring use the 7-amino-3-(1-carboxyethyl)-1-methyl-pyrimido (4,5-c)- pyridazine-4,5(1H; 6H)-dione (AMPPD) co-crystal structure as the source structure. RMSD calculations are used to determine how well specific docking/scoring combinations pose and score the ligand in the pterin site. Surflex with Surflex-Score and Glide with GlideScore are the best overall performers for use in virtual screening against the DHPS target, with neither combination showing statistically significant superiority over the other in enrichment studies or pose selection. Post-docking ligand relaxation and consensus scoring does not improve overall enrichment
drug development
medicine
molecular biology
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established coupled enzymatic assay for kinetic analyses of DHPS activity (coupled to pyrophosphate-dependent phosphofructokinase, aldolase, triosephosphate isomerase, alpha-glycerophosphate dehydrogenase) in presence or absence of activity-modulating compounds
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