Cloned (Comment) | Organism |
---|---|
recombinant expression of N-terminally His-tagged wild-type and mutant enzymes | Mycobacterium tuberculosis |
Crystallization (Comment) | Organism |
---|---|
hanging-drop vapor-diffusion method at 25°C, crystal structures of native enzyme (FBPaseII) at 2.6 A resolution and two active-site protein variants. The variants are complexed with the reaction product fructose 6-phosphate. Presence of a 222 tetramer | Mycobacterium tuberculosis |
purified recombinant wild-type apoform and two active site mutants T84A and T84S in complex with D-fructose 1,6-bisphosphate or D-fructose 6-phosphate (F6P), hanging drop vapor diffusion method, mixing of protein and precipitant solution in a 1:1 ratio, the reservoir solution contains 1.0 M ammonium citrate tribasic, pH 7.0, and 1% PEG 3350, for the apoenzyme, and 2.4 M sodium malonate, pH 6.0, with 1 mM ligand, for the complexed mutant enzyme crystals, X-ray diffraction structure determination and analysis at 2.6 A, 2.3 A, and 2.2 A resolution, respectively, crystal structures of apo MtFBPaseII, the T84A MtFBPase-F6P complex and the T84S MtFBPase-F6P complex are solved by molecular replacement | Mycobacterium tuberculosis |
Protein Variants | Comment | Organism |
---|---|---|
T84A | inactive mutant enzyme | Mycobacterium tuberculosis |
T84A | site-directed mutagenesis, catalytically inactive active-site mutant | Mycobacterium tuberculosis |
T84S | site-directed mutagenesis, active-site mutant, the mutant shows reduced activity compared to wild-type. One Mg2+ ion is found in T84S MtFBPaseII, coordinated to a glycerol molecule and to Asp79, Asp82, and Glu208 near the cleaved 1-phosphate group of the substrate | Mycobacterium tuberculosis |
T84S | lower catalytic activity than wild-type enzyme | Mycobacterium tuberculosis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
2-oxoglutarate | - |
Mycobacterium tuberculosis | |
alpha-ketoglutarate | - |
Mycobacterium tuberculosis | |
citrate | - |
Mycobacterium tuberculosis | |
isocitrate | - |
Mycobacterium tuberculosis | |
malate | - |
Mycobacterium tuberculosis | |
malonate | - |
Mycobacterium tuberculosis | |
additional information | no inhibition by isocitrate | Mycobacterium tuberculosis | |
oxaloacetate | - |
Mycobacterium tuberculosis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required, enzyme-bound | Mycobacterium tuberculosis | |
additional information | the apoenzyme does not contain Mg2+ in the active site, instead Mg2+ is found in helices H8 and H9 in one chain, coordinated to Leu165 N, Gln164 N and Arg161 O and possibly stabilizing this loop. The fructose 6-phosphate-bound structures contain one magnesium ion in a position that is proposed to bind fructose 1,6-bisphosphate, coordinate the 1-phosphate and stabilize the transition state | Mycobacterium tuberculosis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-fructose 1,6-bisphosphate + H2O | Mycobacterium tuberculosis | - |
D-fructose 6-phosphate + phosphate | - |
? | |
D-fructose 1,6-bisphosphate + H2O | Mycobacterium tuberculosis ATCC 25618 | - |
D-fructose 6-phosphate + phosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Mycobacterium tuberculosis | P9WN20 | - |
- |
Mycobacterium tuberculosis | P9WN21 | - |
- |
Mycobacterium tuberculosis ATCC 25618 | P9WN21 | - |
- |
Purification (Comment) | Organism |
---|---|
- |
Mycobacterium tuberculosis |
recombinant N-terminally His-tagged wild-type and mutant enzymes by nickel affinity chromatography and gel filtration | Mycobacterium tuberculosis |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
D-fructose 1,6-bisphosphate + H2O = D-fructose 6-phosphate + phosphate | the proposed mechanism involves two metal ions near the cleavable phosphate: one to stabilize the negative charge on the leaving group and one to coordinate the nucleophilic water | Mycobacterium tuberculosis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-fructose 1,6-bisphosphate + H2O | - |
Mycobacterium tuberculosis | D-fructose 6-phosphate + phosphate | - |
? | |
D-fructose 1,6-bisphosphate + H2O | - |
Mycobacterium tuberculosis ATCC 25618 | D-fructose 6-phosphate + phosphate | - |
? |
Subunits | Comment | Organism |
---|---|---|
homotetramer | - |
Mycobacterium tuberculosis |
homotetramer | a 222 tetramer | Mycobacterium tuberculosis |
More | the constellation of amino-acid residues in the active site of FBPaseII from Mycobacterium tuberculosis (MtFBPaseII) is conserved and is analogous to that described previously for the Escherichia coli enzyme, structure analysis and comparisons, overview | Mycobacterium tuberculosis |
Synonyms | Comment | Organism |
---|---|---|
class II fructose-1,6-bisphosphatase | - |
Mycobacterium tuberculosis |
FBPaseII | - |
Mycobacterium tuberculosis |
GlpX protein | - |
Mycobacterium tuberculosis |
MtFBPaseII | - |
Mycobacterium tuberculosis |
IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
---|---|---|---|---|---|
2 | - |
pH and temperature not specified in the publication | Mycobacterium tuberculosis | citrate | |
2 | - |
versus MtFBPaseII, pH and temperature not specified in the publication | Mycobacterium tuberculosis | citrate | |
3.1 | - |
pH and temperature not specified in the publication | Mycobacterium tuberculosis | alpha-ketoglutarate | |
3.1 | - |
versus MtFBPaseII, pH and temperature not specified in the publication | Mycobacterium tuberculosis | 2-oxoglutarate | |
3.2 | - |
pH and temperature not specified in the publication | Mycobacterium tuberculosis | malonate | |
3.2 | - |
versus MtFBPaseII, pH and temperature not specified in the publication | Mycobacterium tuberculosis | malonate | |
3.3 | - |
pH and temperature not specified in the publication | Mycobacterium tuberculosis | malate | |
3.3 | - |
versus MtFBPaseII, pH and temperature not specified in the publication | Mycobacterium tuberculosis | malate | |
6.4 | - |
pH and temperature not specified in the publication | Mycobacterium tuberculosis | oxaloacetate | |
6.4 | - |
versus MtFBPaseII, pH and temperature not specified in the publication | Mycobacterium tuberculosis | oxaloacetate |
General Information | Comment | Organism |
---|---|---|
evolution | structural comparison of class I versus class II FBPases | Mycobacterium tuberculosis |