Cloned (Comment) | Organism |
---|---|
subcloning in Escherichia coli strain XL1-Blue, recombinant expression of His-tagged wild-type and mutant enzymes in Bacillus subtilis strain WB800 that is deficient in eight extracellular proteases | Paucimonas lemoignei |
Crystallization (Comment) | Organism |
---|---|
purified recombinant His-tagged wild-type enzyme and mutants Y105A, Y105E, Y190E, and S136A, and deletion mutant DELTA202-208 with bound 3-hydroxybutyrate tetramer, hanging drop vapour diffusion method, mixing of 0.002 ml of 10 mg/ml proteinin mM Tris-HCl, pH 8.5, with 0.002 ml of precipitant solution, and equilibration against 0.8 ml of precipitant solution at 16°C, wild-type enzyme and Y190E mutant are crystallized in 0.1 M NaOAc, pH 5.0, 0.2 M MgCl2 and 18% w/v PEG 6000. Mutant Y105A crystals are grown in 0.1 M NaOAc, pH 5.0, 0.2 M NH4Cl and 20% w/v PEG 6000. The DELTA202-208 mutant is crystallized in 0.1 M NaOAc, pH 5.0, 0.2 M LiCl and 15% w/v PEG 6000. Mutant Y105E is crystallized in 0.1 M NaOAc, pH 5.0, 0.2 M LiCl and 18% w/v PEG 6000, mutant S136A is crystallized in 0.1 M NaOAc, pH 5.0, 0.2 M NaCl and 20% w/v PEG 6000, soaking of crystals in ligand solution, X-ray diffraction structure determination and analysis at 1.6-2.0 A resolution | Paucimonas lemoignei |
Protein Variants | Comment | Organism |
---|---|---|
F198E | site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme | Paucimonas lemoignei |
F251E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
F9E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
F9E/S136A | site-directed mutagenesis, inactive mutant | Paucimonas lemoignei |
additional information | construction of a PhaZ7 variant with deletion of residues 202-208. Exchange of surface-exposed amino acids, Y105, Y176, Y189, Y189, W207, that constitute the substrate binding site of the enzyme, by less hydrophobic, hydrophilic or negatively charged residues reduces binding of the enzyme to substrate poly(3-hydroxybutyrate). Modifications of other residues at the enzyme surface, i.e. F9, Y66, Y103, Y124, Y169, Y172, Y173, F198, Y203, Y204, F251, and W252, have no effect on substrate binding | Paucimonas lemoignei |
S136A | site-directed mutagenesis, active site mutation, inactive mutant | Paucimonas lemoignei |
S136A/?W202-V208 | site-directed mutagenesis, inactive mutant | Paucimonas lemoignei |
S136A/W207E | site-directed mutagenesis, inactive mutant | Paucimonas lemoignei |
S136A/W252E | site-directed mutagenesis, inactive mutant | Paucimonas lemoignei |
S136A/Y176E | site-directed mutagenesis, inactive mutant | Paucimonas lemoignei |
S136A/Y189E | site-directed mutagenesis, inactive mutant | Paucimonas lemoignei |
S136A/Y190E | site-directed mutagenesis, inactive mutant | Paucimonas lemoignei |
W202-V208 | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
W207E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
W252E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y103A | site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y103E | site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y105A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y105E | site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y105E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y105E/S136A | site-directed mutagenesis, inactive mutant | Paucimonas lemoignei |
Y105E/S136A/Y189E | site-directed mutagenesis, inactive mutant, no substrate binding | Paucimonas lemoignei |
Y105E/S136A/Y190E | site-directed mutagenesis, inactive mutant, no substrate binding | Paucimonas lemoignei |
Y105E/Y189E | site-directed mutagenesis | Paucimonas lemoignei |
Y105E/Y189E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y105E/Y190E | site-directed mutagenesis | Paucimonas lemoignei |
Y105E/Y190E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y105F | site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y124E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y169E | site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y172A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y173S | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y176E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y189A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y189E | site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y189E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y190A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y190E | site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y190E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y203S | site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y204S | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y66E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Paucimonas lemoignei |
Y66E/S136A | site-directed mutagenesis, inactive mutant | Paucimonas lemoignei |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
poly(3-hydroxybutyrate) + H2O | Paucimonas lemoignei | - |
? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Paucimonas lemoignei | Q939Q9 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Bacillus subtilis strain WB800 by affinity chromatography | Paucimonas lemoignei |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-nitrophenyl octanoate + H2O | - |
Paucimonas lemoignei | 4-nitrophenol + octanoate | - |
? | |
poly(3-hydroxybutyrate) + H2O | - |
Paucimonas lemoignei | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
PhaZ7 | - |
Paucimonas lemoignei |
PHB depolymerase | - |
Paucimonas lemoignei |
poly(3-hydroxybutyrate) depolymerase | - |
Paucimonas lemoignei |
General Information | Comment | Organism |
---|---|---|
additional information | five amino acids, Y105, Y176, Y189, Y189, W207, that constitute the substrate binding site of the enzyme are all located at a single surface-exposed location of the enzyme | Paucimonas lemoignei |