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

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 Tyr105. 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 Tyr103, Tyr172, Tyr173, Tyr203 or Tyr204 to alanine or serine had no phenotype but muteins with substitution of Tyr189, Tyr190 and Tyr105 to alanine showed a lag phase of the in vitro PHB depolymerase reaction. Replacement of Tyr105 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 Tyr105Glu mutant compared to the wild type protein and confirmed that Tyr105 is involved in interaction with the polymeric substrate.