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Literature summary for 3.6.1.1 extracted from
- Archaeal inorganic pyrophosphatase displays robust activity under high-salt conditions and in organic solvents (2016), Appl. Environ. Microbiol., 82, 538-548 .
Application
| Application | Comment | Organism |
|---|---|---|
| synthesis | HvPPA is useful for hydrolyzing diphosphate under conditions of reduced water activity that are a hurdle to current PPA-based technologies | Haloferax volcanii |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene ipp, sequence comparisons and phylogenetic analysis, recombinant expression of N-terminally His6-tagged enzyme in Haloferax volcanii strain H26-pJAM2920 | Haloferax volcanii |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| Sodium fluoride | inhibition kinetics | Haloferax volcanii |  |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | HvPPA displays non-Michaelis-Menten kinetics with a Vmax of 465 U/mg for diphosphate hydrolysis at optimal conditions of 42°C and pH 8.5, and Hill coefficients that indicate cooperative binding to diphosphate and Mg2+ | Haloferax volcanii | |
| 0.26 | - |
diphosphate | recombinant enzyme, pH 8.5, 25°C | Haloferax volcanii | |
| 0.55 | - |
diphosphate | recombinant enzyme, pH 8.5, 42°C | Haloferax volcanii | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | requires Mg2+ or Mn2+, Km at 25°C and pH 8.5 is 13.4 mM. Sigmoidal kinetic profiles indicative of positive cooperative binding are detected for Mg2+ | Haloferax volcanii | |
| Mn2+ | requires Mg2+ or Mn2+ | Haloferax volcanii | |
| NaCl | the enzyme is fully active up to 3 M | Haloferax volcanii | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| diphosphate + H2O | Haloferax volcanii | - |
2 phosphate | - |
? | |
Organic Solvent Stability
| Organic Solvent | Comment | Organism |
|---|---|---|
| additional information | HvPPA is highly negative in surface charge and shows extreme resistance to organic solvents | Haloferax volcanii |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Haloferax volcanii | D4GT97 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant N-terminally His6-tagged enzyme 322fold from Haloferax volcanii strain H26-pJAM2920 to homogeneity by a nickel affinity chromatography, tag cleavage by thrombin, and gel filtration, followed by ultrafiltration | Haloferax volcanii |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| diphosphate + H2O | - |
Haloferax volcanii | 2 phosphate | - |
? | |
| additional information | enzyme HvPPA drives thermodynamically unfavorable reactions to completion under conditions of reduced water activity, development of a coupled assay. HvPPA hydrolyzes the PPi by-product generated in 2 M NaCl by UbaA (a salt-loving noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Malachite green assay. Significant levels of phosphate are detected when UbaA and HvPPA are coupled with ATP and SAMP1 in the reaction, phosphate is not detected when ATP, UbaA, HvPPA, or SAMP1 are omitted from the adenylation assay, and no or poor amounts of phosphate are generated enzymatically when ATP is replaced by other nucleotides (AMP, ADP, AMP-PNP, CTP, GTP, TTP, and UTP) | Haloferax volcanii | ? | - |
- |
|
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homohexamer | 6 * 27000, SDS-PAGE | Haloferax volcanii |
| More | three-dimensional homology modeling suggests HvPPA to have an OB fold consisting of a central beta-barrel structure and alpha-helices associated in a beta1-8-alpha1-beta9-alpha2 topology and to homo-oligomerize into a trimer and/or dimer of trimers | Haloferax volcanii |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| class A type PPA | - |
Haloferax volcanii |
| HVO_0729 | locus name | Haloferax volcanii |
| HvPPA | - |
Haloferax volcanii |
| inorganic pyrophosphatase | - |
Haloferax volcanii |
| IPP | - |
Haloferax volcanii |
| PPA | - |
Haloferax volcanii |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 42 | - |
- |
Haloferax volcanii |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 20 | 60 | recombinant enzyme, activity range, profile overview | Haloferax volcanii |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 65 | - |
purified recombinant enzyme, pH 8.5, half-life is 2 h | Haloferax volcanii |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1050 | - |
diphosphate | recombinant enzyme, pH 8.5, 42°C | Haloferax volcanii | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8.5 | - |
optimum at 25°C | Haloferax volcanii |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | 10.5 | recombinant enzyme, over 50% of maximal activity within this range | Haloferax volcanii |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | hierarchical clustering and three-dimensional (3D) homology modeling reveals that HvPPA is distinct in structure from characterized inorganic diphosphatases, PPAs. HvPPA beongs to the class A type inorganic diphosphatases, PPAs. Evolutionary relationships of archaeal PPAs, overview | Haloferax volcanii |
| additional information | HvPPA is highly negative in surface charge, which explains its extreme resistance to organic solvents. Active site structure comparisons, overview. Three-dimensional homology modeling suggests HvPPA to have an OB fold consisting of a central beta-barrel structure and alpha-helices associated in a beta1-8-alpha1-beta9-alpha2 topology and to homo-oligomerize into a trimer and/or dimer of trimers. Active-site residues of diphosphate hydrolysis are found conserved in HvPPA, including Asp69, which is predicted to provide the carboxylate functional group that performs the nucleophilic attack on the diphosphate substrate when Mg2+ ions are present. The two cysteine residues, Cys24 and Cys85, of HvPPA are found in a Cys-X63-Cys configuration that is highly conserved among haloarchaeal PPAs and distinct from other class A type PPAs | Haloferax volcanii |
| physiological function | hydrolysis of diphosphate byenzyme PPA releases a considerable amount of energy that can drive unfavorable biochemical transformations to completion | Haloferax volcanii |
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