Q-302. Characterization of a Class I Polyhydroxyalkanoate Synthase Isolated from its Native Organism, Ralstonia eutropha
Polyhydroxyalkanoates (PHAs), a class of sustainable polymers, are naturally occurring polyesters produced by a variety of bacteria as carbon storage compounds. PHAs have shown potential as an alternative to petroleum based plastics. The most common PHA found in nature is the homopolymer polyhydroxybutyrate (PHB). PHB can be synthesized by enzymes called polyhydroxyalkanoate synthases, which are divided into four classes based on the monomer/dimer state and the substrate specificity of the enzyme. Class I synthases, such as those present in the bacterium Ralstonia eutropha, are dimers composed of PhaC subunits. PhaC from R. eutropha (PhaCRe) produces short chain length PHAs (PHASCL) from short-chain hydroxyalkanoic acid monomers. The PhaCRe enzyme has been considered a paradigm for Class I synthases. To date, what is known about the PhaCRe enzyme has been characterized from the recombinant enzyme expressed in Escherichia coli. Characterization of PhaCRe, as purified from the native organism has not been explored. We have constructed a gene encoding an epitope tagged PhaCRe and expressed it in recombinant E. coli and, ultimately, in R. eutropha. A "floppy" linker region was introduced between the epitope tag and the PhaC protein, to minimize masking of the epitope by PHB granules or granule-associated proteins. Purification of the resulting protein from R. eutropha results in a highly purified protein preparation. In enzyme activity assays, the recombinant PhaCRe exhibits a brief lag in activity. The native PhaC enzyme exhibits no lag phase, and its activity was measured to be about 10 U/mg protein, with the Km-HBCoA = 129 uM and Vmax-HBCoA = 26.7 U/mg. Absence of the lag phase in activity of the native enzyme is not due to the presence of an interacting protein, as no such factor was purified concomitantly with PhaC in R. eutropha. Using size exclusion chromatography, the recombinant and native enzymes were shown to exist in solution as a mixture of monomer, dimer, and aggregate. The aggregate fraction of the native enzyme preparation displayed ~10X more enzyme activity than the aggregate fraction of the recombinant enzyme. This work represents the first characterization of PhaCRe from its native source.