Literature summary extracted from
Levy, C.; Minnis, D.; Derrick, J.P.
Dihydropteroate synthase from Streptococcus pneumoniae: structure, ligand recognition and mechanism of sulfonamide resistance (2008), Biochem. J., 412, 379-388.
Cloned(Commentary)
EC Number |
Cloned (Comment) |
Organism |
---|
2.5.1.15 |
from Streptococcus pneumoniae for expression in Escherichia coli XL2-Blue |
Streptococcus pneumoniae |
Crystallization (Commentary)
EC Number |
Crystallization (Comment) |
Organism |
---|
2.5.1.15 |
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 |
Streptococcus pneumoniae |
Protein Variants
EC Number |
Protein Variants |
Comment |
Organism |
---|
2.5.1.15 |
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 |
Streptococcus pneumoniae |
2.5.1.15 |
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 |
Streptococcus pneumoniae |
Inhibitors
EC Number |
Inhibitors |
Comment |
Organism |
Structure |
---|
2.5.1.15 |
sulfamethoxazole |
a sulfonamide, KD = 2.3 +/-0.1 microM as revealed by fluorescence spectroscopy |
Streptococcus pneumoniae |
|
Organism
EC Number |
Organism |
UniProt |
Comment |
Textmining |
---|
2.5.1.15 |
Streptococcus pneumoniae |
P59655 |
- |
- |
Purification (Commentary)
EC Number |
Purification (Comment) |
Organism |
---|
2.5.1.15 |
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 |
Streptococcus pneumoniae |
Substrates and Products (Substrate)
EC Number |
Substrates |
Comment Substrates |
Organism |
Products |
Comment (Products) |
Rev. |
Reac. |
---|
2.5.1.15 |
p-aminobenzoic acid + 6-hydroxymethyl-7,8-dihydropterin diphosphate |
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 |
Streptococcus pneumoniae |
diphosphate + 7,8-dihydropteroate |
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 |
? |
|
Synonyms
EC Number |
Synonyms |
Comment |
Organism |
---|
2.5.1.15 |
DHPS |
- |
Streptococcus pneumoniae |
2.5.1.15 |
dihydropteroate synthase |
- |
Streptococcus pneumoniae |