Cloned (Comment) | Organism |
---|---|
expression of His-tagged wild-type PhyK and of mutant H25A PhyK in Escherichia coli strain C41 (DE3) | Klebsiella sp. |
Crystallization (Comment) | Organism |
---|---|
native PhyK and inactive mutant H25A phytase PhyK, the latter with bound sulfate ions, hanging-drop vapor diffusion at 18°C, 5-6 weeks, for the wild-type enzyme: mixing of 6 mg/ml protein in 20 mM sodium acetate (pH 5.0), 50 mm NaCl with an equal volume of 4.0 M sodium formate, for mutant H25A: mixing of 0.0015 ml of protein in 25 mM sodium acetate pH 5.0, 60 mM NaCl and 1 mM tris-(2-carboxyethyl)phosphine with 0.0015 ml of 12% PEG 8000, 0.08 M (NH4)2SO4, 0.1 M sodium acetate and 1.5 mM phytate, 1 week, 22°C, X-ray diffraction structure determination and analysis at 1.7 A resolution, single-wavelength anomalous-diffraction phasing | Klebsiella sp. |
Protein Variants | Comment | Organism |
---|---|---|
H25A | inactive mutant | Klebsiella sp. |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | Klebsiella sp. | phytase ASr1 acts primarily as a scavenger of phosphate groups locked in the phytic acid molecule | ? | - |
? | |
myo-inositol-1,2,3,4,5,6-hexakisphosphate + H2O | Klebsiella sp. | although the PhyK homolog AppA is biochemically characterized as a 6-phytase, a co-crystal structure shows the phytate 3-phosphate as scissile group in a similar position to in the active site of PhyK | myo-inositol-1,2,4,5,6-pentakisphosphate + phosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Klebsiella sp. | Q84CN9 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type PhyK and of mutant H25A PhyK from Escherichia coli strain C41 (DE3) by nickel affinity chromatography | Klebsiella sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | phytase ASr1 acts primarily as a scavenger of phosphate groups locked in the phytic acid molecule | Klebsiella sp. | ? | - |
? | |
additional information | substrate binding structure modelling, all six phosphate groups of phytate are involved in a hydrogen bond network connecting the substrate with PhyK, induced conformational changes upon substrate binding, overview | Klebsiella sp. | ? | - |
? | |
myo-inositol-1,2,3,4,5,6-hexakisphosphate + H2O | although the PhyK homolog AppA is biochemically characterized as a 6-phytase, a co-crystal structure shows the phytate 3-phosphate as scissile group in a similar position to in the active site of PhyK | Klebsiella sp. | myo-inositol-1,2,4,5,6-pentakisphosphate + phosphate | - |
? | |
myo-inositol-1,2,3,4,5,6-hexakisphosphate + H2O | although the PhyK homolog AppA is biochemically characterized as a 6-phytase, a co-crystal structure shows the phytate 3-phosphate as scissile group in a similar position to in the active site of PhyK | Klebsiella sp. | 1D-myo-inositol-1,2,4,5,6-pentakisphosphate + phosphate | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | the ASR1 phytase polypeptide chain is organized into an alpha and an alpha,beta domain, and the active site is located in a positively charged cleft between the domains | Klebsiella sp. |
Synonyms | Comment | Organism |
---|---|---|
ASR1 | - |
Klebsiella sp. |
HAP | - |
Klebsiella sp. |
histidine acid phosphatase | - |
Klebsiella sp. |
More | phytase ASR1 is a member of the histidine-acid-phosphatase family, that shares two conserved active-site motifs, RHGXRXP and HD, and hydrolyzes metal-free phytate with pH optima in the acidic range | Klebsiella sp. |
PhyK | - |
Klebsiella sp. |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
370 | - |
myo-inositol-1,2,3,4,5,6-hexakisphosphate | pH not specified in the publication, temperature not specified in the publication | Klebsiella sp. |
General Information | Comment | Organism |
---|---|---|
additional information | phytase ASR1 is a member of the histidine-acid-phosphatase family, that shares two conserved active-site motifs, RHGXRXP and HD, and hydrolyzes metal-free phytate with pH optima in the acidic range. Histidine-acid-phosphatases share a common two-step mechanism for catalysis. The reaction starts with a nucleophilic attack on the phosphoester bond by a conserved histidine in the long active-site motif. The histidine side chain from the conserved HD motif protonates the leaving group. The second step comprises hydrolysis of the resulting covalent phospho-histidine intermediate. Comparison of substrate binding of Klebsiella sp. ASR1 PhyK with Escherichia coli AppA phytase | Klebsiella sp. |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
7900 | - |
myo-inositol-1,2,3,4,5,6-hexakisphosphate | pH not specified in the publication, temperature not specified in the publication | Klebsiella sp. |