Cloned (Comment) | Organism |
---|---|
from Streptococcus pneumoniae for expression in Escherichia coli XL2-Blue | Streptococcus pneumoniae |
Crystallization (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
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 |
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 | Comment | Organism | Structure |
---|---|---|---|
sulfamethoxazole | a sulfonamide, KD = 2.3 +/-0.1 microM as revealed by fluorescence spectroscopy | Streptococcus pneumoniae |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Streptococcus pneumoniae | P59655 | - |
- |
Purification (Comment) | Organism |
---|---|
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | Comment | Organism |
---|---|---|
DHPS | - |
Streptococcus pneumoniae |
dihydropteroate synthase | - |
Streptococcus pneumoniae |