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Literature summary 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

Cloned(Commentary)

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

Protein Variants

EC Number Protein Variants Comment Organism
3.1.3.8 I15T site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 I15T/R162G site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 I15V/P62S site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 I15Y/P94S site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 K45E site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 L398S/C342G site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 M1T/H146R site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 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
3.1.3.8 T44I/K45E site-directed mutagenesis, the active site loop mutant shows increased activity compared to the wild-type enzyme, hybrid homology modeling Yersinia mollaretii
3.1.3.8 T44M site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 T44R/K45M site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 T44V/K45A site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 T44V/K45D site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 T44V/K45E site-directed mutagenesis, the active site loop mutant shows increased activity compared to the wild-type enzyme, hybrid homology modeling Yersinia mollaretii
3.1.3.8 T44V/K45S site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 T44V/K45W site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 T44V/K45Y site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii
3.1.3.8 Y27C/K45E/K306E site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Yersinia mollaretii

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.1.3.8 1.562
-
 4-methylumbelliferyl phosphate recombinant mutant T44V/K45E, pH 6.6, 37°C Yersinia mollaretii
3.1.3.8 1.598
-
 4-methylumbelliferyl phosphate recombinant mutant T44I/K45E, pH 6.6, 37°C Yersinia mollaretii
3.1.3.8 3.495
-
 4-methylumbelliferyl phosphate recombinant wild-type enzyme, pH 6.6, 37°C Yersinia mollaretii

Natural Substrates/ Products (Substrates)

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

Organism

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

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
3.1.3.8 0.25
-
 recombinant wild-type enzyme, pH 6.6, 37°C Yersinia mollaretii
3.1.3.8 1.68
-
 recombinant mutant T44I/K45E, pH 6.6, 37°C Yersinia mollaretii
3.1.3.8 1.79
-
 recombinant mutant T44V/K45E, pH 6.6, 37°C Yersinia mollaretii

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.1.3.8 4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii 4-methylumbelliferone + phosphate
-
 ?
3.1.3.8 4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii CNY 7263 4-methylumbelliferone + phosphate
-
 ?
3.1.3.8 4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii DSM 18520 4-methylumbelliferone + phosphate
-
 ?
3.1.3.8 4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii CIP 103324 4-methylumbelliferone + phosphate
-
 ?
3.1.3.8 4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii ATCC 43969 4-methylumbelliferone + phosphate
-
 ?
3.1.3.8 4-methylumbelliferyl phosphate + H2O phytase activity detection using 4-methylumbelliferyl phosphate as substrate Yersinia mollaretii WAIP 204 4-methylumbelliferone + phosphate
-
 ?
3.1.3.8 myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
3.1.3.8 myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii CNY 7263 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
3.1.3.8 myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii DSM 18520 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
3.1.3.8 myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii CIP 103324 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
3.1.3.8 myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii ATCC 43969 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?
3.1.3.8 myo-inositol hexakisphosphate + H2O
-
 Yersinia mollaretii WAIP 204 1D-myo-inositol 1,2,4,5,6-pentakisphosphate + phosphate
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.1.3.8 AppA
-
 Yersinia mollaretii
3.1.3.8 histidine acid phosphatase
-
 Yersinia mollaretii
3.1.3.8 YmPh
-
 Yersinia mollaretii

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.1.3.8 37
-
 assay at Yersinia mollaretii

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.1.3.8 5.33
-
 4-methylumbelliferyl phosphate recombinant wild-type enzyme, pH 6.6, 37°C Yersinia mollaretii
3.1.3.8 10.18
-
 4-methylumbelliferyl phosphate recombinant mutant T44V/K45E, pH 6.6, 37°C Yersinia mollaretii
3.1.3.8 10.42
-
 4-methylumbelliferyl phosphate recombinant mutant T44I/K45E, pH 6.6, 37°C Yersinia mollaretii

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.1.3.8 5.6
-
-
 Yersinia mollaretii

pH Range

EC Number pH Minimum pH Maximum Comment Organism
3.1.3.8 additional information
-
 pH-dependence of wild-type and mutant enzymes, overview Yersinia mollaretii
3.1.3.8 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

EC Number General Information Comment Organism
3.1.3.8 evolution the enzyme belongs to the histidine acid phosphatases Yersinia mollaretii
3.1.3.8 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.1.3.8 1.53
-
 4-methylumbelliferyl phosphate recombinant wild-type enzyme, pH 6.6, 37°C Yersinia mollaretii
3.1.3.8 6.2
-
 4-methylumbelliferyl phosphate recombinant mutant T44I/K45E, pH 6.6, 37°C Yersinia mollaretii
3.1.3.8 6.52
-
 4-methylumbelliferyl phosphate recombinant mutant T44V/K45E, pH 6.6, 37°C Yersinia mollaretii