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Literature summary for 3.5.2.2 extracted from
- Characterization and soluble expression of D-hydantoinase from Pseudomonas fluorescens for the synthesis of D-amino acids (2016), Appl. Biochem. Biotechnol., 179, 1-15 .
Application
| Application | Comment | Organism |
|---|---|---|
| synthesis | recombinant D-PfHYD can potentially be applied in the synthesis of D-amino acids | Pseudomonas fluorescens |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene hyd, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis and tree, recombinant expression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3)-pET28-hyd/pGRO7, mostly in insoluble form. Five sets of molecular chaperones are coexpressed with D-PfHYD in Escherichia coli, including pGRO7 (GroES-GroEL), pKJE7 (DnaK-DnaJ-GrpE), pTF16 (Tigger), pGRO7-KJE8 (GroES-GroEL & DnaK-DnaJ-GrpE), and pGRO7-TF2, to improve the method. Coexpression of the chaperone GroES-GroEL is most efficient in improving the soluble expression of D-PfHYD | Pseudomonas fluorescens |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| EDTA | addition of metal chelating agent has little inhibitory effect on the activity of enzyme D-PfHYD | Pseudomonas fluorescens |  |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 4.42 | - |
isobutylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 4.64 | - |
indolmethylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 7.52 | - |
4-hydroxyphenylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 7.63 | - |
isopropylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 8.57 | - |
2-chlorobenzenehydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 9.49 | - |
phenylmethylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Fe2+ | activates 20% at 1 mM | Pseudomonas fluorescens | |
| Fe3+ | activates 20% at 1 mM | Pseudomonas fluorescens | |
| Mn2+ | activates 20% at 1 mM | Pseudomonas fluorescens | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 215000 | - |
recombinant N-terminally His-tagged enzyme, gel filtration | Pseudomonas fluorescens |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| isobutylhydantoin + H2O | Pseudomonas fluorescens | 98.7 % enantiomeric excess (ee), isolation yield of 78.3%, and substrate to biocatalyst ratio of 15.6 in recombinant Escherichia coli | N-carbamoyl-D-alpha-isoleucine | - |
? | |
| isobutylhydantoin + H2O | Pseudomonas fluorescens CGMCC 1.1802 | 98.7 % enantiomeric excess (ee), isolation yield of 78.3%, and substrate to biocatalyst ratio of 15.6 in recombinant Escherichia coli | N-carbamoyl-D-alpha-isoleucine | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Pseudomonas fluorescens | S5MPT0 | - |
- |
| Pseudomonas fluorescens CGMCC 1.1802 | S5MPT0 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged enzyme 3.91fold from Escherichia coli strain BL21(DE3) by nickel affinity chromatography to homogeneity | Pseudomonas fluorescens |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 5.34 | - |
crude enzyme, pH 9.0, 45°C, substrate isobutylhydantoin | Pseudomonas fluorescens |
| 20.9 | - |
purified recombinant enzyme, pH 9.0, 45°C | Pseudomonas fluorescens |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2-chlorobenzenehydantoin + H2O | - |
Pseudomonas fluorescens | ? | - |
? | |
| 2-chlorobenzenehydantoin + H2O | - |
Pseudomonas fluorescens CGMCC 1.1802 | ? | - |
? | |
| 4-hydroxyphenylhydantoin + H2O | - |
Pseudomonas fluorescens | N-carbamoyl-D-hydroxyphenylglycine | - |
? | |
| 4-hydroxyphenylhydantoin + H2O | - |
Pseudomonas fluorescens CGMCC 1.1802 | N-carbamoyl-D-hydroxyphenylglycine | - |
? | |
| indolmethylhydantoin + H2O | - |
Pseudomonas fluorescens | tryptophan | - |
? | |
| isobutylhydantoin + H2O | - |
Pseudomonas fluorescens | N-carbamoyl-D-alpha-isoleucine | - |
? | |
| isobutylhydantoin + H2O | 98.7 % enantiomeric excess (ee), isolation yield of 78.3%, and substrate to biocatalyst ratio of 15.6 in recombinant Escherichia coli | Pseudomonas fluorescens | N-carbamoyl-D-alpha-isoleucine | - |
? | |
| isobutylhydantoin + H2O | - |
Pseudomonas fluorescens CGMCC 1.1802 | N-carbamoyl-D-alpha-isoleucine | - |
? | |
| isobutylhydantoin + H2O | 98.7 % enantiomeric excess (ee), isolation yield of 78.3%, and substrate to biocatalyst ratio of 15.6 in recombinant Escherichia coli | Pseudomonas fluorescens CGMCC 1.1802 | N-carbamoyl-D-alpha-isoleucine | - |
? | |
| isopropylhydantoin + H2O | - |
Pseudomonas fluorescens | N-carbamoyl-D-valine | - |
? | |
| additional information | purified D-PfHYD prefers aliphatic to aromatic 5'-monosubstituted hydantoins. 5'-Monosubstituted hydantoins give N-carbamoyl-D-alpha-amino acids | Pseudomonas fluorescens | ? | - |
- |
|
| additional information | purified D-PfHYD prefers aliphatic to aromatic 5'-monosubstituted hydantoins. 5'-Monosubstituted hydantoins give N-carbamoyl-D-alpha-amino acids | Pseudomonas fluorescens CGMCC 1.1802 | ? | - |
- |
|
| phenylmethylhydantoin + H2O | - |
Pseudomonas fluorescens | phenylhydantoic acid | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homotetramer | 4 * 55000, recombinant N-terminally His-tagged enzyme, SDS-PAGE, 4 * 54400, about, sequence calculation | Pseudomonas fluorescens |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| D-HYD | - |
Pseudomonas fluorescens |
| D-hydantoinase | - |
Pseudomonas fluorescens |
| D-PfHYD | - |
Pseudomonas fluorescens |
| dihydropyrimidase/hydantoinase | - |
Pseudomonas fluorescens |
| HYD | - |
Pseudomonas fluorescens |
| hydantoinase | - |
Pseudomonas fluorescens |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 60 | - |
recombinant enzyme | Pseudomonas fluorescens |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | 60 | maximal activity of D-PfHYD at about 60°C, 29.4% and 62.0% of maximal activity at 30°C and 45°C, respectively, over 60% of maximal activity at 50-70°C | Pseudomonas fluorescens |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.37 | - |
2-chlorobenzenehydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 1.43 | - |
indolmethylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 2.91 | - |
phenylmethylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 74.2 | - |
4-hydroxyphenylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 77.3 | - |
isopropylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 391 | - |
isobutylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 9 | - |
recombinant enzyme | Pseudomonas fluorescens |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 6 | 10.5 | the enzyme displays highest activity at pH 9.0 and shows over 90% of maximal activity at pH 8.5-9.5, and over 50% at pH 7.0-10.5. Below pH 7.5, the activity decreases sharply, 5% of maximal activity at pH 6.0 | Pseudomonas fluorescens |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | several hydrolases have been reported with hydantoin hydrolysis activity, including amidohydrolase, dihydropyrimidase/hydantoinase, allantoinase, and dihydroorotase, which belong to the cyclic amidase superfamily containing 22 potential subfamilies. According to the phylogenetic tree, D-PfHYD displays a close relationship to dihydropyrimidase/hydantoinase, whereas it is distinct from allantoinase (ALN, AllB, LhyD, and FeAllB) and dihydroorotase (PyrC, Ura4). Phylogenetic analysis and tree, overview | Pseudomonas fluorescens |
| additional information | the catalytic residues are conserved in D-PfHYD, including His59, His61, Lys150, His183, His239, and Asp 316, which participate in the hydrolysis reaction by interaction with metal ions and hydantoin substrates through the carbamate group, amide nitrogen and carbonyl oxygen | Pseudomonas fluorescens |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.043 | - |
2-chlorobenzenehydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 0.31 | - |
phenylmethylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 0.31 | - |
indolmethylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 9.87 | - |
4-hydroxyphenylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 10.13 | - |
isopropylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
| 88.46 | - |
isobutylhydantoin | pH 9.0, 45°C, recombinant enzyme | Pseudomonas fluorescens | |
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