Application | Comment | Organism |
---|---|---|
drug development | the structure of the effector site of LmFBPase shows different structural features compared with human FBPases, thereby offering a potential and species-specific drug target | Leishmania major |
Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli BL21(DE3) Gold strain | Leishmania major |
Crystallization (Comment) | Organism |
---|---|
purified enzyme LmFBPase as apoenzyme, and LmFBPase complexed with Mn2+ and product phosphate or substrate fructose 1,6-bisphosphate, or LmFBPase complexed with its allosteric inhibitor AMP, hanging drop vapor diffusion technique, mixing of 0.0015 ml of 3.8 mg/ml of protein in 20 mM TEA, 5 mM MgCl2, 50 mM KCl, 10% glycerol, 1 mM fructose 1,6-bisphosphate, pH 7.0, with 0.0015 ml reservoir solution containing 20% w/v PEG 3350, 0.05 M citric acid, and 0.05 M Bis-Tris propane, pH 5.0, 17°C, 3 days, for ligand-bound enzyme crystals, mixing of 0.0015 ml of 4 mg/ml protein in 20 mM TEA, 5 mM MgCl2, 50 mM KCl, 10% glycerol, 1 mM substrate/product, 10 mM MnCl2, or 2 mM AMP, pH 7.0-7.2, with 0.0015 ml of reservoir solution containing 20% w/v PEG 3350, 0.05 M citric acid, and 0.05 M Bis-Tris propane, pH 6.0, 3 weeks, 17°C, X-ray diffraction structure determination and analysis at 2.41-2.9 A resolution, molecular replacement using the pig liver FBPase structure (PDB ID 5FBP) as template | Leishmania major |
vapor diffusion using the hanging-drop technique | Leishmania major |
General Stability | Organism |
---|---|
the stabilization effect of the substrate fructose-1,6-bisphosphate is not eliminated by AMP, but the stabilizing effects are additive. The catalytic product fructose-6-phosphate also shows a stabilization effect on LmFBPase, but not as significant as the substrate | Leishmania major |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
AMP | allosteric inhibitor. AMP binding triggers a rearrangement of hydrogen bonds across the large and small interfaces. Retraction of the effector loop required for AMP binding releases the side chain of His23 from the dimer-dimer interface. This is coupled with a flip of the side chain of Arg48 which ties down the key catalytic dynamic loop in a disengaged conformation and also locks the tetramer in an inactive rotated T-state; AMP binds to an effector site about 30 A distant from the active site and acts as an allosteric inhibitor, the crystal structure of LmFBPase complexed with its allosteric inhibitor AMP shows an inactive form of the tetramer, in which the dimer pairs are rotated by 18° relative to each other, revealing an allosteric mechanism in which AMP binding triggers a rearrangement of hydrogen bonds across the large and small interfaces. Retraction of the effector loop required for AMP binding releases the side chain of His23 from the dimer-dimer interface. This is coupled with a flip of the side chain of Arg48 which ties down the key catalytic dynamic loop in a disengaged conformation and also locks the tetramer in an inactive rotated T-state. AMP inhibits the catalytic activity of LmFBPase without eliminating the substrate binding | Leishmania major | |
Ca2+ | similar inhibition constant as Mn2+ | Leishmania major | |
fructose-2,6-bisphosphate | fructose 2,6-bisphosphate binds at the active site, and shows a synergistic inhibitory effect with AMP | Leishmania major | |
Mn2+ | thermostability of LmFBPase is increased by more than 9°C in the presence of Mn2+ | Leishmania major |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | activity assays show that the substrate D-fructose 1,6-bisphosphate binds to the enzyme with positive cooperativity, Michaelis-Menten plot | Leishmania major | |
0.0198 | - |
D-fructose 1,6-bisphosphate | pH 8.0, 37°C | Leishmania major |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required, Km is 1.2 mM | Leishmania major | |
additional information | metal binding structure of the enzyme, overview | Leishmania major |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-fructose 1,6-bisphosphate + H2O | Leishmania major | - |
D-fructose 6-phosphate + phosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Leishmania major | O97193 | - |
- |
Purification (Comment) | Organism |
---|---|
- |
Leishmania major |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
D-fructose 1,6-bisphosphate + H2O = D-fructose 6-phosphate + phosphate | metal-dependent reaction mechanism, allosteric mechanism of the enzyme, overview | Leishmania major |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-fructose 1,6-bisphosphate + H2O | - |
Leishmania major | D-fructose 6-phosphate + phosphate | - |
? | |
additional information | the higher Km for D-fructose 1,6-bisphosphate of LmFBPase compared to the Escherichia coli enzyme may be explained by a sequence difference at the active site, with Tyr221 replaced by Asn221 resulting in the loss of a direct hydrogen bond with substrate D-fructose 1,6-bisphosphate. Binding structure analysis of substrate and product, overview | Leishmania major | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
homotetramer | the apoform of enzyme LmFBPase is a homotetramer in which the dimer of dimers adopts a planar conformation with disordered dynamic loops | Leishmania major |
homotetramer | the dimer of dimers adopts a planar conformation with disordered dynamic loops | Leishmania major |
More | enzyme structure analysis and structure-function relationship, overview. Not only interactions across both the small and the large interfaces, but also interactions between diagonal chains are formed. Two independent, but identical symmetry-related clusters of hydrogen bonds, each lying across the 2fold symmetry axis of the tetramer, are formed by Asn195 and Arg48. The rearrangement of these eight hydrogen bonds that stabilize the core of the homotetramer in the planar R-state regulates the R to T switching mechanism | Leishmania major |
Synonyms | Comment | Organism |
---|---|---|
fructose-1,6-bisphosphatase | - |
Leishmania major |
LmFBPase | - |
Leishmania major |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Leishmania major |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
8 | 56.5 | temperature midpoint for the protein unfolding transition of enzyme LmFBPase is 56.5°C, thermostability in the presence of the substrate fructose-2,6-bisphosphate is highly increased for LmFBPase with a DELTATm of about 8°C. The stabilization effect of the substrate is not eliminated by AMP, but the stabilizing effects are additive. Thermostability of LmFBPase is increased by more than 9°C in the presence of Mn2+ | Leishmania major |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
8 | - |
D-fructose 1,6-bisphosphate | pH 8.0, 37°C | Leishmania major |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Leishmania major |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0019 | - |
fructose-2,6-bisphosphate | pH 8.0, 37°C | Leishmania major | |
0.0443 | - |
Mn2+ | pH 8.0, 37°C | Leishmania major | |
0.0638 | - |
AMP | pH 8.0, 37°C | Leishmania major |
General Information | Comment | Organism |
---|---|---|
drug target | potential drug target against Leishmania parasites | Leishmania major |
evolution | structures of Leishmania fructose-1,6-bisphosphatase (FBPase) reveal species-specific differences in the mechanism of allosteric inhibition of FBPases | Leishmania major |
metabolism | gluconeogenic enzyme | Leishmania major |
additional information | the apoform of enzyme LmFBPase is a homotetramer in which the dimer of dimers adopts a planar conformation with disordered dynamic loops. The structure of LmFBPase, complexed with manganese and the catalytic product phosphate, shows the dynamic loops locked into the active sites. The dynamic loop (residues 52-71), which has been shown to be catalytically important in mammalian FBPases, is in a similar position in LmFBPase but shows functionally important sequence differences and has two insertions (Tyr57 and Gln61) compared with mammalian and bacterial sequences. Enzyme structure analysis and structure-function relationship, modeling, overview. Conformational variability of the FBPase active site | Leishmania major |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
404 | - |
D-fructose 1,6-bisphosphate | pH 8.0, 37°C | Leishmania major |