Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 3.5.2.2 extracted from

  • Zhang, X.; Niu, L.; Shi, Y.; Yuan, J.
    The flexibility of the non-conservative region at the C terminus of D-hydantoinase from Pseudomonas putida YZ-26 is extremely limited (2008), Appl. Biochem. Biotechnol., 144, 237-247.
    View publication on PubMed
Search for more literature for 3.5.2.2

Application

Application Comment Organism
industry production of optically pure D-amino acids that are key intermediates in the synthesis of commercial products such as beta-lactam semisynthetic antibiotics, peptides, hormones, pyretroids and pesticides Pseudomonas putida

Cloned(Commentary)

Cloned (Comment) Organism
a series of mutants of the enzyme with the C-terminal residues either deleted or substituted are prepared, expression in Escherichia coli Pseudomonas putida
expressed in Escherichia coli BL21 (DE3) cells Pseudomonas putida

Protein Variants

Protein Variants Comment Organism
DELTA474-479 C-terminally truncated mutant, expressed in the form of random aggregates without any activity Pseudomonas putida
DELTA475-479 C-terminally truncated mutant, dissociated into the monomeric state, the activity is largely retained Pseudomonas putida
DELTA476-479 C-terminally truncated mutant, dissociated into the monomeric state, the activity is largely retained Pseudomonas putida
DELTA477-479 C-terminally truncated mutant, dissociated into the monomeric state, the activity is largely retained Pseudomonas putida
DELTA478-479 C-terminally truncated mutant, dissociated into the monomeric state, the activity is largely retained Pseudomonas putida
additional information the truncated mutants P478, P477, P476, and P475 are dissociated into the monomeric state, but their activities are largely retained (86.7-57.0% of wild type activity) Pseudomonas putida
additional information the flexibility of the non-conservative region at the C-terminus is quite limited implying that the intact enzyme structure is essential for enzyme activity Pseudomonas putida
additional information several truncated mutants (P478, P477, P476, and P475) are dissociated into the monomeric state as well, but their activities are largely retained, the relative activities of mutants is about twice as that of the wild type enzyme at pH 6.0 and pH 10.0 Pseudomonas putida
R474 mutant is expressed in the form of random aggregates without any activity Pseudomonas putida
R479A mutant is expressed in the form of random aggregates without any activity Pseudomonas putida
R479A expressed in the form of random aggregates without any activity Pseudomonas putida
R479A the mutant is expressed in the form of random aggregates without any activity, the relative activity of mutant is about twice as that of the wild type enzyme at pH 6.0 and pH 10.0 Pseudomonas putida
R479D dissociates into the monomeric state but 78.2% retains activity of the wild type enzyme Pseudomonas putida
R479D dissociated into the monomeric state, activity is largely retained Pseudomonas putida
R479D the C-terminal-substituted enzyme is dissociated into the monomeric state, but the activity is largely retained, the relative activity of mutant is about twice as that of the wild type enzyme at pH 6.0 and pH 10.0 Pseudomonas putida

Inhibitors

Inhibitors Comment Organism Structure
Zn2+ 60% increase of activity at 1 mM Pseudomonas putida

Metals/Ions

Metals/Ions Comment Organism Structure
Mn2+ 96% residual activity at 1 mM Pseudomonas putida
Mn2+ compared to wild-type without metal ions, 1 mM Mn2+ enhances the activity up to 200% for wild-type enzyme P479 and to 400–500% for truncates mutants P477, P476, P475 and mutant R479D Pseudomonas putida
additional information binding sites of Zn2+ and Mn2+ may locate at different regions on the enzyme Pseudomonas putida
Zn2+ compared to wild-type without metal ions, 1 mM Zn2+ attenuates the enzymatic activity down to 60% for wild-type enzyme P479 and to more than 90% for mutants R479D and truncated mutants P477, P476 and P475 Pseudomonas putida
Zn2+ Zn2+-binding enzyme Pseudomonas putida

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
54000
-
 SDS-PAGE Pseudomonas putida
54000
-
 molecular weight of the monomer enzyme and its mutants evaluated by SDS-PAGE, molecular weight of their native forms are determined by gel filtration Pseudomonas putida
110000
-
 SDS-PAGE Pseudomonas putida

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
additional information Pseudomonas putida catalyzes the hydrolysis of 5-monosubstituted hydantoin to enantionmeric N-carbamoyl-amino acids ?
-
 ?
additional information Pseudomonas putida YZ-26 catalyzes the hydrolysis of 5-monosubstituted hydantoin to enantionmeric N-carbamoyl-amino acids ?
-
 ?

Organism

Organism UniProt Comment Textmining
Pseudomonas putida
-
-
-
Pseudomonas putida YZ-26
-
-
-

Purification (Commentary)

Purification (Comment) Organism
purification of mutants by a two-step chromatography, an ion exchange chromatography following by a hydrophobic chromatography Pseudomonas putida
Q-Sepharose column chromatography, ammonium sulfate precipitation, and phenyl-Sepharose column chromatography Pseudomonas putida
Q-Sepharose Fast Flow chromatography and Phenyl-Sepharose Fast Flow column chromatography Pseudomonas putida

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 0.01 U/ml cells, mutant DELTA474-479 Pseudomonas putida
additional information
-
 0.06 U/ml cells, mutant R479A Pseudomonas putida
additional information
-
 1.32 U/ml cells, mutant DELTA477-479 Pseudomonas putida
additional information
-
 1.33 U/ml cells, mutant DELTA478-479 Pseudomonas putida
additional information
-
 1.40 U/ml cells, mutant DELTA475-479 Pseudomonas putida
additional information
-
 1.48 U/ml cells, mutant DELTA476-479 Pseudomonas putida
additional information
-
 1.73 U/ml cells, mutant R479D Pseudomonas putida
additional information
-
 2.12 U/ml cells, mutant DELTA479 Pseudomonas putida
additional information
-
 no activity in mutant P474, free enzyme Pseudomonas putida
additional information
-
 no activity in mutant R479A, free enzyme Pseudomonas putida
additional information
-
 only the last four residues can be deleted without significantly affecting the enzyme activity Pseudomonas putida
6.6
-
 mutant P477, free enzyme Pseudomonas putida
8.1
-
 mutant P476, free enzyme Pseudomonas putida
8.41
-
 purified mutant enzyme R479D, at 37°C Pseudomonas putida
8.41
-
 mutant R479D, free enzyme Pseudomonas putida
9.32
-
 mutant P478, ifree enzyme Pseudomonas putida
10.75
-
 purified wild type enzyme, at 37°C Pseudomonas putida
10.75
-
 mutant P479, free enzyme Pseudomonas putida
613
-
 mutant P475, free enzyme Pseudomonas putida

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
hydantoin + H2O
-
 Pseudomonas putida N-carbamoyl glycine
-
 ?
hydantoin + H2O
-
 Pseudomonas putida YZ-26 N-carbamoyl glycine
-
 ?
hydantoin + H2O
-
 Pseudomonas putida N-carbamoylglycine
-
 ?
hydantoin + H2O
-
 Pseudomonas putida YZ-26 N-carbamoylglycine
-
 ?
additional information catalyzes the hydrolysis of 5-monosubstituted hydantoin to enantionmeric N-carbamoyl-amino acids Pseudomonas putida ?
-
 ?
additional information catalyzes the hydrolysis of 5-monosubstituted hydantoin to enantionmeric N-carbamoyl-amino acids Pseudomonas putida YZ-26 ?
-
 ?

Subunits

Subunits Comment Organism
dimer
-
 Pseudomonas putida
dimer wild-type P479, determined by gel filtration Pseudomonas putida
homodimer 2 * 54000, SDS-PAGE Pseudomonas putida
monomer mutant enzymes R479D, P478, P477, P476, P475, and P474, determined by size-exclusion chromatography Pseudomonas putida

Synonyms

Synonyms Comment Organism
D-hydantoinase
-
 Pseudomonas putida
DHP
-
 Pseudomonas putida
P479
-
 Pseudomonas putida

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Pseudomonas putida

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
 The thermal stability of mutant entyme P479 is higher than that of all mutants under the same conditions. The oligomeric D-hydantoinase may be less flexible and resist the thermal inactivation. The monomer enzyme has less these interaction forces that may affect its stability under the externally environmental factors. Pseudomonas putida

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 mutants Pseudomonas putida
8 9
-
 Pseudomonas putida

pH Range

pH Minimum pH Maximum Comment Organism
7 8 wild-type enzyme, 100% activity Pseudomonas putida

pH Stability

pH Stability pH Stability Maximum Comment Organism
additional information
-
 pH stability of mutant enzymes R479D and C-terminally truncated mutants P477, P476, and P475 is significantly higher than that of the wild-type enzyme at both acidic and basic sides. Pseudomonas putida
additional information
-
 The influence of pH, either on the wild-type enzyme or on mutants, is close to the optimal pH from pH 8.0 to 9.0 Pseudomonas putida
additional information
-
 The relative activity of mutants is about twice as that of the wild-type enzyme at pH 6.0 and pH 10.0 Pseudomonas putida