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

Abstract

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-Ser 136 -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 Ser 136 Cys) 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 : His 47 Ala, Ser 136 Ala, Asp 242 Ala, Asp 242 Asn, His 306 Ala, indicating the importance of these amino acids for activity. Replacement of Ser 136 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 Asp 256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser 136 -Met-Gly) to a lipase box consensus sequence (Gly 134 -His-Ser 136 -Met-Gly) or to the PHB depolymerase box consensus sequence (Gly 134 -Leu 135 -Ser 136 -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 His 47 , Ser 136 , Asp 242 and His 306 .

Acyltransferases in Bacteria

“…The essential roles of Ser102, His251, and Asp223 were confirmed by the observation that a replacement of each of these amino acids with alanine diminished PhaG activity (269). This type of catalytic triad with serine is found in enzymes belonging to the serine hydrolase superfamily, which act, for example, as lipases, PHA depolymerases, serine hydrolases, (thio-)esterases, or fatty acid or PK synthases (233,(276)(277)(278)(279)(280)(281). As mentioned above, the carbonyl carbon atom of the acyl group is covalently attached to the serine hydroxyl group of the enzyme and is subsequently released or, in case of PhaG, transesterified to ACP or CoA.…”

Identification and Biochemical Evidence of a Medium-Chain-Length Polyhydroxyalkanoate Depolymerase in the Bdellovibrio bacteriovorus Predatory Hydrolytic Arsenal

“…These biobased monomers are important chiral starting scaffolds in material, fine chemical, pharmaceutical, and medical industries (7,46). Several bacterial extracellular PHA depolymerases have been identified and characterized so far (4,26,28,29). PHA depolymerases belong to the ␣/␤-hydrolase fold family and have a catalytic triad (serine-histidine-aspartic acid) as the active site, with the exception of the intracellular scl-PHA depolymerase superfamily (29).…”

“…The ability to degrade extracellular scl-PHA is more widespread among bacteria than the ability to degrade mcl-PHA. Thus, many extracellular scl-PHA depolymerases have been characterized in depth over the last decade, and a considerable number of genes have been identified (1,3,4,24,26,29,40). The prototype of extracellular mcl-PHA depolymerases is that of Pseudomonas fluorescens GK13 (here PhaZ GK13 ) (21,26,52).…”

Biochemical Evidence That phaZ Gene Encodes a Specific Intracellular Medium Chain Length Polyhydroxyalkanoate Depolymerase in Pseudomonas putida KT2442

“…Identification of the Products Released from PHA after Enzymatic Hydrolysis-For the identification of the PhaZ hydrolysis products, four reaction mixtures were subjected to enzymatic hydrolysis in parallel with 250 g of P(HO-co-HX)w at different reaction times (1,12,30, and 360 min). The first three mixtures were developed in the presence of 1.8 g of PhaZ as a standard turbidimetric assay.…”