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Literature summary for 3.6.1.1 extracted from

  • Huang, H.; Patskovsky, Y.; Toro, R.; Farelli, J.D.; Pandya, C.; Almo, S.C.; Allen, K.N.; Dunaway-Mariano, D.
    Divergence of structure and function in the haloacid dehalogenase enzyme superfamily: Bacteroides thetaiotaomicron BT2127 is an inorganic pyrophosphatase (2011), Biochemistry, 50, 8937-8949.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.6.1.1

Cloned(Commentary)

Cloned (Comment) Organism
phylogenetic analysis, expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3) Bacteroides thetaiotaomicron

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant His-tagged wild-type and mutant BT2127 variants, sitting-drop vapor diffusion method, X-ray diffraction structure determination and analysis at Bacteroides thetaiotaomicron

Protein Variants

Protein Variants Comment Organism
D11N site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron
D13A site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron
D13N site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron
E47A site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron
E47D site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron
E47N site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron
E47Q site-directed mutagenesis, altered kinetics compared to the wild-type enzyme Bacteroides thetaiotaomicron
H23A site-directed mutagenesis, altered kinetics compared to the wild-type enzyme Bacteroides thetaiotaomicron
K147A site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron
K79A site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron
M20A site-directed mutagenesis, altered kinetics compared to the wild-type enzyme Bacteroides thetaiotaomicron
M20K site-directed mutagenesis, altered kinetics compared to the wild-type enzyme Bacteroides thetaiotaomicron
M20L site-directed mutagenesis, altered kinetics compared to the wild-type enzyme Bacteroides thetaiotaomicron
N172A site-directed mutagenesis, altered kinetics compared to the wild-type enzyme Bacteroides thetaiotaomicron
Q117A site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron
R49A site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron
S115A site-directed mutagenesis, altered kinetics compared to the wild-type enzyme Bacteroides thetaiotaomicron
S19A site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron
S80A site-directed mutagenesis, altered kinetics compared to the wild-type enzyme Bacteroides thetaiotaomicron
T113A site-directed mutagenesis, altered kinetics compared to the wild-type enzyme Bacteroides thetaiotaomicron
T50A site-directed mutagenesis, altered kinetics compared to the wild-type enzyme Bacteroides thetaiotaomicron
Y76F site-directed mutagenesis, inactive mutant Bacteroides thetaiotaomicron

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information wild-type and mutant BT2127 steady-state kinetics, overview Bacteroides thetaiotaomicron
0.32
-
 diphosphate pH 7.5, 25°C, recombinant wild-type BT2127 Bacteroides thetaiotaomicron

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required, functions in catalysis and binding structure, overview Bacteroides thetaiotaomicron
additional information the metal binding residues are Asp171, Asn172, and Glu47 Bacteroides thetaiotaomicron

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
diphosphate + H2O Bacteroides thetaiotaomicron in vivo function as an inorganic diphosphatase. Substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain, specifically by residues Tyr76 and Glu47 2 phosphate
-
 ?
diphosphate + H2O Bacteroides thetaiotaomicron ATCC 29148 in vivo function as an inorganic diphosphatase. Substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain, specifically by residues Tyr76 and Glu47 2 phosphate
-
 ?

Organism

Organism UniProt Comment Textmining
Bacteroides thetaiotaomicron Q8A5V9
-
-
Bacteroides thetaiotaomicron ATCC 29148 Q8A5V9
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 (DE3) by anion exchange chromatography, hydrophobic interaction chromatography, and nickel affinity chromatography Bacteroides thetaiotaomicron

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
diphosphate + H2O in vivo function as an inorganic diphosphatase. Substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain, specifically by residues Tyr76 and Glu47 Bacteroides thetaiotaomicron 2 phosphate
-
 ?
diphosphate + H2O structure-function analysis, overview. BT2127 conserves the His23-Lys79 diad, and in both the cap-open and -closed conformations, the Asp13 side chain is in the same conformation, engaged in a hydrogen bond with linker residue Ser15 Bacteroides thetaiotaomicron 2 phosphate
-
 ?
diphosphate + H2O in vivo function as an inorganic diphosphatase. Substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain, specifically by residues Tyr76 and Glu47 Bacteroides thetaiotaomicron ATCC 29148 2 phosphate
-
 ?
diphosphate + H2O structure-function analysis, overview. BT2127 conserves the His23-Lys79 diad, and in both the cap-open and -closed conformations, the Asp13 side chain is in the same conformation, engaged in a hydrogen bond with linker residue Ser15 Bacteroides thetaiotaomicron ATCC 29148 2 phosphate
-
 ?
additional information BT2127 substrate specificity profile, overview Bacteroides thetaiotaomicron ?
-
 ?
additional information BT2127 substrate specificity profile, overview Bacteroides thetaiotaomicron ATCC 29148 ?
-
 ?

Subunits

Subunits Comment Organism
More structure modeling and structure-function analysis, overview. BT2127 conserves the His23-Lys79 diad, and in both the cap-open and -closed conformations, the Asp13 side chain is in the same conformation, engaged in a hydrogen bond with linker residue Ser15 Bacteroides thetaiotaomicron

Synonyms

Synonyms Comment Organism
BT2127
-
 Bacteroides thetaiotaomicron
inorganic pyrophosphatase
-
 Bacteroides thetaiotaomicron

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Bacteroides thetaiotaomicron

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.6
-
 diphosphate pH 7.5, 25°C, recombinant wild-type BT2127 Bacteroides thetaiotaomicron

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Bacteroides thetaiotaomicron

General Information

General Information Comment Organism
evolution enzyme BT2127 is a member of the haloalkanoate dehalogenase superfamily, HADSF Bacteroides thetaiotaomicron
additional information the catalytic residues are Asp11, Asp13, Thr113, and Lys147, structure-guided site-directed mutagenesis coupled with kinetic analysis of the mutant enzymes identifies the residues required for catalysis, substrate binding, and domain-domain association Bacteroides thetaiotaomicron

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
89
-
 diphosphate pH 7.5, 25°C, recombinant wild-type BT2127 Bacteroides thetaiotaomicron