Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei

The recently discovered extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser₁₃₆-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids. In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800. The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser₁₃₆Cys) were expressed and efficiently secreted by B. subtilis as shown by Western blots of cell-free culture fluid proteins. No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His₄₇Ala, Ser₁₃₆Ala, Asp₂₄₂Ala, Asp₂₄₂Asn, His₃₀₆Ala, indicating the importance of these amino acids for activity. Replacement of Ser₁₃₆ by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine. Alterations of Asp₂₅₆ to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser₁₃₆-Met-Gly) to a lipase box consensus sequence (Gly₁₃₄-His-Ser₁₃₆-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly₁₃₄-Leu₁₃₅-Ser₁₃₆-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity. In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His₄₇, Ser₁₃₆, Asp₂₄₂ and His₃₀₆.