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Literature summary for 2.7.6.3 extracted from

  • Shaw, G.X.; Li, Y.; Shi, G.; Wu, Y.; Cherry, S.; Needle, D.; Zhang, D.; Tropea, J.E.; Waugh, D.S.; Yan, H.; Ji, X.
    Structural enzymology and inhibition of the bi-functional folate pathway enzyme HPPK-DHPS from the biowarfare agent Francisella tularensis (2014), FEBS J., 281, 4123-4137.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.7.6.3

Application

Application Comment Organism
drug development the enzyme is a target for antibiotic development Francisella tularensis

Cloned(Commentary)

Cloned (Comment) Organism
gene folK, sequence comparisons, recombinant expression of His6-tagged and maltose-binding-protein-tagged enzyme in Escherichia coli strain BL21-CodonPlus(DE3)-RIL Francisella tularensis

Crystallization (Commentary)

Crystallization (Comment) Organism
purified enzyme in complex with inhibitor HP-26, sitting drop vapour diffusion method, mixing of 300 nl 11 mg/ml protein in 25 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM TCEP, and saturated HP-26, with 300 nl well solution containing 28% w/v poly(ethylene glycol) monomethyl ether 2000, and 100 mM Bis-Tris, pH 6.5, X-ray diffraction structure determination and analysis at 1.7 A resolution Francisella tularensis

Inhibitors

Inhibitors Comment Organism Structure
2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic acid (2-(2-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethanesulfonyl]-thylcarbamoyl)-ethyl)-amide 2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic acid (2-(2-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethanesulfonyl]-thylcarbamoyl)-ethyl)-amide, structure of the enzyme complex with HP-26. HP-26 has significant isozyme selectivity. HP-26 is an excellent lead for developing therapeutic agents for tularemia Francisella tularensis
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'-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, structure of the enzyme complex with HP-18. HP-18 has significant isozyme selectivity Francisella tularensis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state kinetic analysis Francisella tularensis
0.0018
-
 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pH 8.3, 30°C, recombinant enzyme Francisella tularensis
0.019
-
 ATP pH 8.3, 30°C, recombinant enzyme Francisella tularensis

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Francisella tularensis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine Francisella tularensis
-
 AMP + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
-
 ?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine Francisella tularensis SCHU S4
-
 AMP + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
-
 ?
additional information Francisella tularensis the bifunctional enzyme comprises the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) activity and dihydropteroate synthase (DHPS) activity, EC 2.5.1.15, the latter is very low due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid ?
-
 ?
additional information Francisella tularensis SCHU S4 the bifunctional enzyme comprises the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) activity and dihydropteroate synthase (DHPS) activity, EC 2.5.1.15, the latter is very low due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid ?
-
 ?

Organism

Organism UniProt Comment Textmining
Francisella tularensis Q5NGA7 gene folK, single copy gene
-
Francisella tularensis SCHU S4 Q5NGA7 gene folK, single copy gene
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged and maltose-binding-protein-tagged enzyme from Escherichia coli strain BL21-CodonPlus(DE3)-RIL by nickel affinity chromatography and ultrafiltration, the tags are cleaved off by TEV protease, followed by gel filtration Francisella tularensis

Reaction

Reaction Comment Organism Reaction ID
ATP + 6-hydroxymethyl-7,8-dihydropterin = AMP + 6-hydroxymethyl-7,8-dihydropterin diphosphate ordered kinetic mechanism, with Mg2+-dependent ATP binding followed by rapid addition of 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine Francisella tularensis
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
-
 Francisella tularensis AMP + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
-
 ?
ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine
-
 Francisella tularensis SCHU S4 AMP + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate
-
 ?
additional information the bifunctional enzyme comprises the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) activity and dihydropteroate synthase (DHPS) activity, EC 2.5.1.15, the latter is very low due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid Francisella tularensis ?
-
 ?
additional information the bifunctional enzyme comprises the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) activity and dihydropteroate synthase (DHPS) activity, EC 2.5.1.15, the latter is very low due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid Francisella tularensis SCHU S4 ?
-
 ?

Synonyms

Synonyms Comment Organism
2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase
-
 Francisella tularensis
2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase/dihydropteroate synthase
-
 Francisella tularensis
6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase
-
 Francisella tularensis
FtHPPK-DHPS
-
 Francisella tularensis
HPPK
-
 Francisella tularensis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Francisella tularensis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.17
-
 2-Amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pH 8.3, 30°C, recombinant enzyme Francisella tularensis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.3
-
 assay at Francisella tularensis

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Francisella tularensis

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.0024
-
 2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic acid (2-(2-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethanesulfonyl]-thylcarbamoyl)-ethyl)-amide pH 8.3, 30°C, recombinant enzyme Francisella tularensis
0.085
-
 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 pH 8.3, 30°C, recombinant enzyme Francisella tularensis

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.0051
-
 pH 8.3, 30°C, recombinant enzyme Francisella tularensis 2-amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic acid (2-(2-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethanesulfonyl]-thylcarbamoyl)-ethyl)-amide
0.18
-
 pH 8.3, 30°C, recombinant enzyme Francisella tularensis 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