BRENDA - Enzyme Database

Structures of the mycobacterium tuberculosis glpx protein (Class II fructose-1,6-bisphosphatase) Implications for the active oligomeric state, catalytic mechanism and citrate inhibition

Wolf, N.; Gutka, H.; Movahedzadeh, F.; Abad-Zapatero, C.; Acta Crystallogr. Sect. D 74, 321-331 (2018)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
3.1.3.11
recombinant expression of N-terminally His-tagged wild-type and mutant enzymes
Mycobacterium tuberculosis
Crystallization (Commentary)
EC Number
Crystallization
Organism
3.1.3.11
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
Engineering
EC Number
Amino acid exchange
Commentary
Organism
3.1.3.11
T84A
site-directed mutagenesis, catalytically inactive active-site mutant
Mycobacterium tuberculosis
3.1.3.11
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
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
3.1.3.11
2-oxoglutarate
-
Mycobacterium tuberculosis
3.1.3.11
citrate
-
Mycobacterium tuberculosis
3.1.3.11
isocitrate
-
Mycobacterium tuberculosis
3.1.3.11
malate
-
Mycobacterium tuberculosis
3.1.3.11
malonate
-
Mycobacterium tuberculosis
3.1.3.11
oxaloacetate
-
Mycobacterium tuberculosis
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
3.1.3.11
Mg2+
required, enzyme-bound
Mycobacterium tuberculosis
3.1.3.11
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/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
3.1.3.11
D-fructose 1,6-bisphosphate + H2O
Mycobacterium tuberculosis
-
D-fructose 6-phosphate + phosphate
-
-
?
3.1.3.11
D-fructose 1,6-bisphosphate + H2O
Mycobacterium tuberculosis ATCC 25618
-
D-fructose 6-phosphate + phosphate
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
3.1.3.11
Mycobacterium tuberculosis
P9WN21
-
-
3.1.3.11
Mycobacterium tuberculosis ATCC 25618
P9WN21
-
-
Purification (Commentary)
EC Number
Commentary
Organism
3.1.3.11
recombinant N-terminally His-tagged wild-type and mutant enzymes by nickel affinity chromatography and gel filtration
Mycobacterium tuberculosis
Reaction
EC Number
Reaction
Commentary
Organism
3.1.3.11
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 and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.1.3.11
D-fructose 1,6-bisphosphate + H2O
-
749502
Mycobacterium tuberculosis
D-fructose 6-phosphate + phosphate
-
-
-
?
3.1.3.11
D-fructose 1,6-bisphosphate + H2O
-
749502
Mycobacterium tuberculosis ATCC 25618
D-fructose 6-phosphate + phosphate
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
3.1.3.11
homotetramer
a 222 tetramer
Mycobacterium tuberculosis
3.1.3.11
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
IC50 Value
EC Number
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
3.1.3.11
2
-
versus MtFBPaseII, pH and temperature not specified in the publication
Mycobacterium tuberculosis
citrate
3.1.3.11
3.1
-
versus MtFBPaseII, pH and temperature not specified in the publication
Mycobacterium tuberculosis
2-oxoglutarate
3.1.3.11
3.2
-
versus MtFBPaseII, pH and temperature not specified in the publication
Mycobacterium tuberculosis
malonate
3.1.3.11
3.3
-
versus MtFBPaseII, pH and temperature not specified in the publication
Mycobacterium tuberculosis
malate
3.1.3.11
6.4
-
versus MtFBPaseII, pH and temperature not specified in the publication
Mycobacterium tuberculosis
oxaloacetate
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
3.1.3.11
recombinant expression of N-terminally His-tagged wild-type and mutant enzymes
Mycobacterium tuberculosis
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
3.1.3.11
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
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
3.1.3.11
T84A
site-directed mutagenesis, catalytically inactive active-site mutant
Mycobacterium tuberculosis
3.1.3.11
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
IC50 Value (protein specific)
EC Number
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
3.1.3.11
2
-
versus MtFBPaseII, pH and temperature not specified in the publication
Mycobacterium tuberculosis
citrate
3.1.3.11
3.1
-
versus MtFBPaseII, pH and temperature not specified in the publication
Mycobacterium tuberculosis
2-oxoglutarate
3.1.3.11
3.2
-
versus MtFBPaseII, pH and temperature not specified in the publication
Mycobacterium tuberculosis
malonate
3.1.3.11
3.3
-
versus MtFBPaseII, pH and temperature not specified in the publication
Mycobacterium tuberculosis
malate
3.1.3.11
6.4
-
versus MtFBPaseII, pH and temperature not specified in the publication
Mycobacterium tuberculosis
oxaloacetate
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
3.1.3.11
2-oxoglutarate
-
Mycobacterium tuberculosis
3.1.3.11
citrate
-
Mycobacterium tuberculosis
3.1.3.11
isocitrate
-
Mycobacterium tuberculosis
3.1.3.11
malate
-
Mycobacterium tuberculosis
3.1.3.11
malonate
-
Mycobacterium tuberculosis
3.1.3.11
oxaloacetate
-
Mycobacterium tuberculosis
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
3.1.3.11
Mg2+
required, enzyme-bound
Mycobacterium tuberculosis
3.1.3.11
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/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
3.1.3.11
D-fructose 1,6-bisphosphate + H2O
Mycobacterium tuberculosis
-
D-fructose 6-phosphate + phosphate
-
-
?
3.1.3.11
D-fructose 1,6-bisphosphate + H2O
Mycobacterium tuberculosis ATCC 25618
-
D-fructose 6-phosphate + phosphate
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
3.1.3.11
recombinant N-terminally His-tagged wild-type and mutant enzymes by nickel affinity chromatography and gel filtration
Mycobacterium tuberculosis
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.1.3.11
D-fructose 1,6-bisphosphate + H2O
-
749502
Mycobacterium tuberculosis
D-fructose 6-phosphate + phosphate
-
-
-
?
3.1.3.11
D-fructose 1,6-bisphosphate + H2O
-
749502
Mycobacterium tuberculosis ATCC 25618
D-fructose 6-phosphate + phosphate
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
3.1.3.11
homotetramer
a 222 tetramer
Mycobacterium tuberculosis
3.1.3.11
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
General Information
EC Number
General Information
Commentary
Organism
3.1.3.11
evolution
structural comparison of class I versus class II FBPases
Mycobacterium tuberculosis
General Information (protein specific)
EC Number
General Information
Commentary
Organism
3.1.3.11
evolution
structural comparison of class I versus class II FBPases
Mycobacterium tuberculosis