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

Abstract

Polyhydroxyalkanoates (PHAs) can be catabolized by many microorganisms using intra-or extracellular PHA depolymerases. Most of our current knowledge of these intracellular enzyme-coding genes comes from the analysis of short chain length PHA depolymerases, whereas medium chain length PHA (mcl-PHA) intracellular depolymerization systems still remained to be characterized. The phaZ gene of some Pseudomonas putida strains has been identified only by mutagenesis and complementation techniques as putative intracellular mcl-PHA depolymerase. However, none of their corresponding encoded PhaZ enzymes have been characterized in depth. In this study the PhaZ depolymerase from P. putida KT2442 has been purified and biochemically characterized after its overexpression in Escherichia coli. To facilitate these studies we have developed a new and very sensitive radioactive method for detecting PHA hydrolysis in vitro. We have demonstrated that PhaZ is an intracellular depolymerase that is located in PHA granules and that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. We have modeled the three-dimensional structure of PhaZ complexed with a 3-hydroxyoctanoate dimer. Using this model, we found that the enzyme appears to be built up from a core ␣/␤ hydrolase-type domain capped with a lid structure with an active site containing a catalytic triad buried near the connection between domains. All these data constitute the first biochemical characterization of PhaZ and allow us to propose this enzyme as the paradigmatic representative of intracellular endo/ exo-mcl-PHA depolymerases.

Polyhydroxyalkanoate Granules Are Complex Subcellular Organelles (Carbonosomes)

“…In P. putida, one of the two acyl-CoA synthetase (in fusion with GFP) was localized in vivo at PHA granules (87). This finding was in agreement with previous findings that up to seven enzymatic activities were identified in isolated PHA granules in P. putida (5,14,102): aside from PHA synthase and PHA depolymerase acyl-CoA synthetase, acyl-CoA dehydrogenase, enoyl-CoA hydratase, hydroxyacyl-CoA reductase, and ketoacyl-CoA reductase were reported. Another important finding was the identification of a protein with regulatory function (PhaR) at the PHB granule surface in R. eutropha by Western blotting and immunogold labeling in thin sections (see above) (73).…”

“…Interestingly, the gene coding for the iPHA MCL depolymerase (phaZ) is located between two copies of PHA MCL synthase genes (phaC1 and phaC2) in all investigated PHA MCLaccumulating bacteria. Recently, iPHA depolymerase activity could be demonstrated in vitro by use of radiolabeled substrates (nPHA MCL ) or by pH stat (nPHA SCL ), respectively (5,16). iPHB depolymerases of PHA SCL -accumulating bacteria were not described before 2000, but in the first 5 years of this century no fewer than seven putative iPHB depolymerases and two 3HB oligomer hydrolases have been postulated for R. eutropha (1, 23, 44-46, 71, 88, 123).…”

Poly(3-Hydroxybutyrate) (PHB) Depolymerase PhaZa1 Is Involved in Mobilization of Accumulated PHB in Ralstonia eutropha H16

“…The literature contains frequent reports of putative iPHB depolymerases (iPhaZs) (1,5,8,32,40,43) or mediumchain-length iPHA (iPHA MCL ) depolymerases (3,4,13). However, a convincing in vitro assay system for iPhaZs does not exist, and unfortunately, researchers have used differently prepared substrates for assays (6,15).…”

Development of a New Strategy for Production of Medium-Chain-Length Polyhydroxyalkanoates by Recombinant Escherichia coli via Inexpensive Non-Fatty Acid Feedstocks

“…The genome of Pseudomonas putida KT2440 is fully sequenced, and several genes involved with PHA synthesis have been identified (1,14). Two PHA synthase genes, phaC1 and phaC2, which are separated by the PHA depolymerase gene phaZ, are characterized as class II PHA synthases (3,17). These synthases generally prefer 3-hydroxyacyl-coenzyme A (CoA) with chain lengths of 6 to 14 carbon atoms as substrates to produce MCL-PHA polymers (20).…”