Tyrosine 105 of Paucimonas lemoignei PHB depolymerase PhaZ7 is essential for polymer binding

摘要

The 3-dimensional structure of the Paucimonas lemoignei poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ7 has significant similarity to Bacillus subtilis lipase LipA but differs from the latter by the presence of an additional domain. Analysis of this lid-like domain revealed the presence of many hydrophobic amino acid residues including Tyr_(105). In this study we constructed His-tag fusions of PhaZ7 for simplified purification and investigated the effect of amino acid exchange of eight tyrosine codons of the lid-like domain. Exchanges of Tyr_(103), Tyr_(172), Tyr_(173), Tyr_(203) or Tyr_(204) to alanine or serine had no phenotype but muteins with substitution of Tyr_(189), Tyr_(190) and Tyr_(105) to alanine showed a lag phase of the in vitro PHB depolymerase reaction. Replacement of Tyr_(105) by glutamate further increased the lag phase. Binding assays of the purified PHB depolymerase proteins with the natural substrate, native PHB granules, revealed a significantly reduced binding ability of the Tyr_(105)Glu mutant compared to the wild type protein and confirmed that Tyrios is involved in interaction with the polymeric substrate.