Literature summary for 3.1.3.11 extracted from

Yuan, M.; Vasquez-Valdivieso, M.G.; McNae, I.W.; Michels, P.A.M.; Fothergill-Gilmore, L.A.; Walkinshaw, M.D.
Structures of Leishmania fructose-1,6-bisphosphatase reveal species-specific differences in the mechanism of allosteric inhibition (2017), J. Mol. Biol., 429, 3075-3089 .

Application

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(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli BL21(DE3) Gold strain	Leishmania major

Crystallization (Commentary)

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

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

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

Inhibitors	Comment	Organism
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 [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

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/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Leishmania major	O97193	-	-

Purification (Commentary)

Purification (Comment)	Organism
-	Leishmania major

Reaction

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	a

Substrates and Products (Substrate)

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

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

Synonyms	Comment	Organism
fructose-1,6-bisphosphatase	-	Leishmania major
LmFBPase	-	Leishmania major

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Leishmania major

Temperature Stability [°C]

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 [1/s]

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

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Leishmania major

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
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

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 [mM/s]

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