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.
Braaz
Reinhard
R
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70550 Stuttgart, Germany.
Handrick
Rene
R
Jendrossek
Dieter
D
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics*; D008049:Lipase / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D001426:Bacterial Proteins; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C438011:PHAZ7 protein, Paucimonas lemoignei; C077226:poly(3-hydroxyalkanoic acid) depolymerase; D008049:Lipase