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Literature summary for 3.6.1.1 extracted from

  • Pratt, A.C.; Dewage, S.W.; Pang, A.H.; Biswas, T.; Barnard-Britson, S.; Cisneros, G.A.; Tsodikov, O.V.
    Structural and computational dissection of the catalytic mechanism of the inorganic pyrophosphatase from Mycobacterium tuberculosis (2015), J. Struct. Biol., 192, 76-87 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli Mycobacterium tuberculosis

Crystallization (Commentary)

Crystallization (Comment) Organism
purified enzyme bound to catalytic metals, to substrate diphosphate, and to one or two inorganic phosphate ions, hanging drop vapour diffusion method, mixing of 0.001 ml of 14 mg/ml protein in 40 mM Tris-HCl, pH 8.0, and 100 mM NaCl, with 0.001 ml of reservoir solution containing 1.65 M NaKHPO4, 100 mM HEPES, pH 7.75, and 2 mM CaCl2, xthe crystals of Mtb PPiase in complex with two phosphate ions are obtained with the reservoir solution composed of 1.6 M KH2PO4, 100 mM HEPES, pH 7.75, and 2 mM CaCl2, and the crystals of Mtb PPiase in complex with one phosphate are obtained with the reservoir solution containing 1.57 M NaKHPO4, 100 mM HEPES pH 7.75 and 2 mM MnCl2, 22°C, X-ray diffraction structure determination and analysis at 1.85-3.30 A resolution, molecular replacement with the structure of the Mtb PPiase-Mg2+ complex as a search model Mycobacterium tuberculosis
purified enzyme, X-ray diffraction structure determination and analysis Escherichia coli

Protein Variants

Protein Variants Comment Organism
D54N site-directed mutagenesis, the mutant behaves similar to the wild-type enzyme Mycobacterium tuberculosis
D57N site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to wild-type Mycobacterium tuberculosis
D89N site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to wild-type Mycobacterium tuberculosis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten steady-state kinetic analysis Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
diphosphate + H2O Escherichia coli
-
2 phosphate
-
r
diphosphate + H2O Mycobacterium tuberculosis
-
2 phosphate
-
r

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
-
-
Mycobacterium tuberculosis P9WI55
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinand His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography, gel filtration, and ultrafiltration Mycobacterium tuberculosis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
diphosphate + H2O
-
Escherichia coli 2 phosphate
-
r
diphosphate + H2O
-
Mycobacterium tuberculosis 2 phosphate
-
r
additional information usage of the malachite green assay. The location and the conformation of the PPi in all 12 active sites are nearly identical, whereas the location and the occupancy of a Ca2+ at position M1 (most commonly coordinating both Asp57 and Asp89) as well as the conformation of the side chain of Asp89 and, to a lesser extent of Asp57, are somewhat variable among the different active sites, substrate binding structures, detailed overview Mycobacterium tuberculosis ?
-
-

Synonyms

Synonyms Comment Organism
Mtb PPiase
-
Mycobacterium tuberculosis
PPIase
-
Escherichia coli
PPIase
-
Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Mycobacterium tuberculosis

General Information

General Information Comment Organism
additional information structure comparisons of substrate-bound enzymes from Mycobacterum tuberculosis and Escherichia coli, quantum mechanics/molecular mechanics (QM/MM) analysis. Mtb PPiase exhibits significant structural differences from the well characterized Escherichia coli PPiase in the vicinity of the bound diphosphate substrate. In Mtb PPiase, Asp89, rather than Asp54 as in Escherichia coli PPiase, can abstract a proton from a water molecule to activate it for a nucleophilic attack on the diphosphate substrate, catalytic reaction mechanism, detailed overview. Structure-function analysis. Asp54 does not play a major catalytic role, and another residue (e.g. Asp89) acts as a catalytic base Mycobacterium tuberculosis
additional information structure comparisons of substrate-bound enzymes from Mycobacterum tuberculosis and Escherichia coli, quantum mechanics/molecular mechanics (QM/MM) analysis. Mycobacterum tuberculosis PPiase exhibits significant structural differences from the well characterized Escherichia coli PPiase in the vicinity of the bound diphosphate substrate. In Escherichia coli PPiase, Asp54 , rather than Asp89 as in Mycobacterum tuberculosis PPiase, can abstract a proton from a water molecule to activate it for a nucleophilic attack on the diphosphate substrate, catalytic reaction mechanism, and structure-function analysis, overview Escherichia coli