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Literature summary extracted from

  • Jendrossek, D.; Hermawan, S.; Subedi, B.; Papageorgiou, A.C.
    Biochemical analysis and structure determination of Paucimonas lemoignei poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ7 muteins reveal the PHB binding site and details of substrate-enzyme interactions (2013), Mol. Microbiol., 90, 649-664.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.1.1.75 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 (Commentary)

EC Number Crystallization (Comment) Organism
3.1.1.75 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

EC Number Protein Variants Comment Organism
3.1.1.75 F198E site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 F251E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 F9E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 F9E/S136A site-directed mutagenesis, inactive mutant Paucimonas lemoignei
3.1.1.75 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
3.1.1.75 S136A site-directed mutagenesis, active site mutation, inactive mutant Paucimonas lemoignei
3.1.1.75 S136A/?W202-V208 site-directed mutagenesis, inactive mutant Paucimonas lemoignei
3.1.1.75 S136A/W207E site-directed mutagenesis, inactive mutant Paucimonas lemoignei
3.1.1.75 S136A/W252E site-directed mutagenesis, inactive mutant Paucimonas lemoignei
3.1.1.75 S136A/Y176E site-directed mutagenesis, inactive mutant Paucimonas lemoignei
3.1.1.75 S136A/Y189E site-directed mutagenesis, inactive mutant Paucimonas lemoignei
3.1.1.75 S136A/Y190E site-directed mutagenesis, inactive mutant Paucimonas lemoignei
3.1.1.75 W202-V208 site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 W207E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 W252E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y103A site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y103E site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y105A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y105E site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y105E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y105E/S136A site-directed mutagenesis, inactive mutant Paucimonas lemoignei
3.1.1.75 Y105E/S136A/Y189E site-directed mutagenesis, inactive mutant, no substrate binding Paucimonas lemoignei
3.1.1.75 Y105E/S136A/Y190E site-directed mutagenesis, inactive mutant, no substrate binding Paucimonas lemoignei
3.1.1.75 Y105E/Y189E site-directed mutagenesis Paucimonas lemoignei
3.1.1.75 Y105E/Y189E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y105E/Y190E site-directed mutagenesis Paucimonas lemoignei
3.1.1.75 Y105E/Y190E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y105F site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y124E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y169E site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y172A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y173S site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y176E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y189A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y189E site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y189E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y190A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y190E site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y190E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y203S site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y204S site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y66E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Paucimonas lemoignei
3.1.1.75 Y66E/S136A site-directed mutagenesis, inactive mutant Paucimonas lemoignei

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.1.1.75 poly(3-hydroxybutyrate) + H2O Paucimonas lemoignei
-
 ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.1.1.75 Paucimonas lemoignei Q939Q9
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.1.1.75 recombinant His-tagged wild-type and mutant enzymes from Bacillus subtilis strain WB800 by affinity chromatography Paucimonas lemoignei

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.1.1.75 4-nitrophenyl octanoate + H2O
-
 Paucimonas lemoignei 4-nitrophenol + octanoate
-
 ?
3.1.1.75 poly(3-hydroxybutyrate) + H2O
-
 Paucimonas lemoignei ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.1.1.75 PhaZ7
-
 Paucimonas lemoignei
3.1.1.75 PHB depolymerase
-
 Paucimonas lemoignei
3.1.1.75 poly(3-hydroxybutyrate) depolymerase
-
 Paucimonas lemoignei

General Information

EC Number General Information Comment Organism
3.1.1.75 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