An extra large insertion in the polyhydroxyalkanoate synthase from Delftia acidovorans DS-17: its deletion effects and relation to cellular proteolysis

The polyhydroxyalkanoate (PHA) synthase (PhaC(Da)) from Delftia acidovorans DS-17 (formerly Comamonas acidovorans) has a unique large insertion consisting of 40 amino acid residues in the alpha/beta hydrolase fold region. In order to examine whether this insertion is necessary for enzyme function, we generated a mutant gene where the nucleotides encoding the insertion sequence were deleted [phaC(Da)del(342-381)]. The ability of the mutant PhaC(Da) lacking the insertion sequence to produce PHA in recombinant Escherichia coli JM109 was compared with that of wild-type PhaC(Da). The results revealed that the mutant enzyme had approximately one fourth the activity of the wild-type enzyme. However, there was no significant difference in PHA content accumulated in cells harboring either the mutant PhaC(Da) or wild-type PhaC(Da) nor were there any differences in the molecular masses of the produced polymers. Therefore, we have concluded that the characteristic insertion is not indispensable for PHA synthesis. Also, slight cellular proteolysis in E. coli was found specifically for wild-type PhaC(Da) by Western blot analysis. This result prompted us to further examine the proteolytic stability of PhaC(Da) in D. acidovorans. Consequently, it has been suggested that the insertion region of PhaC(Da) is susceptible to cellular proteolysis during accumulation of PHA.