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Literature summary for 3.1.3.8 extracted from

  • Koerfer, G.; Novoa, C.; Kern, J.; Balla, E.; Gruetering, C.; Davari, M.D.; Martinez, R.; Vojcic, L.; Schwaneberg, U.
    Directed evolution of an acid Yersinia mollaretii phytase for broadened activity at neutral pH (2018), Appl. Microbiol. Biotechnol., 102, 9607-9620 .
    View publication on PubMed
Search for more literature for 3.1.3.8

Cloned(Commentary)

Cloned (Comment) Organism
gene appA, random YmPh mutant libraries generation and screening, recombinant expression in Escherichia coli strain BL21 Gold (DE3)lacIQ Yersinia mollaretii

Protein Variants

Protein Variants Comment Organism
I15T site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
I15T/R162G site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
I15V/P62S site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
I15Y/P94S site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
K45E site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
L398S/C342G site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
M1T/H146R site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
additional information directed evolution identifies the key positions T44 and K45 for increased YmPh activity at neutral pH. Both positions are located in the active site loop of the phytase and have a synergistic effect on activity with a broadened pH spectrum. Kinetic characterization of the improved variants, YmPh-M10 (T44I/K45E) and YmPh-M16 (T44V/K45E), show up to sevenfold increased specific activity and up to 2.2fold reduced KM at pH 6.6 under screening conditions compared to Yersinia mollaretii phytase wild-type (YmPhWT). pH-Dependence of wild-type and mutant enzymes, overview Yersinia mollaretii
T44I/K45E site-directed mutagenesis, the active site loop mutant shows increased activity compared to the wild-type enzyme, hybrid homology modeling Yersinia mollaretii
T44M site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
T44R/K45M site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
T44V/K45A site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
T44V/K45D site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
T44V/K45E site-directed mutagenesis, the active site loop mutant shows increased activity compared to the wild-type enzyme, hybrid homology modeling Yersinia mollaretii
T44V/K45S site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
T44V/K45W site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
T44V/K45Y site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
Y27C/K45E/K306E site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.562
-
 4-methylumbelliferyl phosphate recombinant mutant T44V/K45E, pH 6.6, 37°C Yersinia mollaretii
1.598
-
 4-methylumbelliferyl phosphate recombinant mutant T44I/K45E, pH 6.6, 37°C Yersinia mollaretii
3.495
-
 4-methylumbelliferyl phosphate recombinant wild-type enzyme, pH 6.6, 37°C Yersinia mollaretii

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
myo-inositol hexakisphosphate + H2O Yersinia mollaretii
-
 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
myo-inositol hexakisphosphate + H2O Yersinia mollaretii CNY 7263
-
 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
myo-inositol hexakisphosphate + H2O Yersinia mollaretii DSM 18520
-
 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
myo-inositol hexakisphosphate + H2O Yersinia mollaretii CIP 103324
-
 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
myo-inositol hexakisphosphate + H2O Yersinia mollaretii ATCC 43969
-
 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
myo-inositol hexakisphosphate + H2O Yersinia mollaretii WAIP 204
-
 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?

Organism

Organism UniProt Comment Textmining
Yersinia mollaretii C4S833
-
-
Yersinia mollaretii ATCC 43969 C4S833
-
-
Yersinia mollaretii CIP 103324 C4S833
-
-
Yersinia mollaretii CNY 7263 C4S833
-
-
Yersinia mollaretii DSM 18520 C4S833
-
-
Yersinia mollaretii WAIP 204 C4S833
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.25
-
 recombinant wild-type enzyme, pH 6.6, 37°C Yersinia mollaretii
1.68
-
 recombinant mutant T44I/K45E, pH 6.6, 37°C Yersinia mollaretii
1.79
-
 recombinant mutant T44V/K45E, pH 6.6, 37°C Yersinia mollaretii

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii 4-methylumbelliferone + phosphate
-
 ?
4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii CNY 7263 4-methylumbelliferone + phosphate
-
 ?
4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii DSM 18520 4-methylumbelliferone + phosphate
-
 ?
4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii CIP 103324 4-methylumbelliferone + phosphate
-
 ?
4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii ATCC 43969 4-methylumbelliferone + phosphate
-
 ?
4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii WAIP 204 4-methylumbelliferone + phosphate
-
 ?
myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii CNY 7263 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii DSM 18520 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii CIP 103324 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii ATCC 43969 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii WAIP 204 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?

Synonyms

Synonyms Comment Organism
AppA
-
 Yersinia mollaretii
histidine acid phosphatase
-
 Yersinia mollaretii
YmPh
-
 Yersinia mollaretii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Yersinia mollaretii

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.33
-
 4-methylumbelliferyl phosphate recombinant wild-type enzyme, pH 6.6, 37°C Yersinia mollaretii
10.18
-
 4-methylumbelliferyl phosphate recombinant mutant T44V/K45E, pH 6.6, 37°C Yersinia mollaretii
10.42
-
 4-methylumbelliferyl phosphate recombinant mutant T44I/K45E, pH 6.6, 37°C Yersinia mollaretii

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5.6
-
-
 Yersinia mollaretii

pH Range

pH Minimum pH Maximum Comment Organism
additional information
-
 pH-dependence of wild-type and mutant enzymes, overview Yersinia mollaretii
5.6 7.4 the activity of the wild-tyype enzyme YmPhWT decreases drastically by 14fold from pH 5.6 to pH 7.4 Yersinia mollaretii

General Information

General Information Comment Organism
evolution the enzyme belongs to the histidine acid phosphatases Yersinia mollaretii
additional information homology structure modeling of Yersinia mollaretii phytase using the Yersinia kristensenii phytase apo-form structure (PDB ID 4ARV), Hafnia alvei phytase in complex with tartrate (PDB ID 4ARU), Hafnia alvei phytase in complex with myo-inositol hexakissulphate (PDB ID 4ARO), Hafnia alvei phytase apo-form (PDB ID 4ARS), and Escherichia coli phytase with bound phytate in the active site (PDB ID 1DKQ) as templates, location of active site loop Yersinia mollaretii

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.53
-
 4-methylumbelliferyl phosphate recombinant wild-type enzyme, pH 6.6, 37°C Yersinia mollaretii
6.2
-
 4-methylumbelliferyl phosphate recombinant mutant T44I/K45E, pH 6.6, 37°C Yersinia mollaretii
6.52
-
 4-methylumbelliferyl phosphate recombinant mutant T44V/K45E, pH 6.6, 37°C Yersinia mollaretii