Active Intermediates of Polyhydroxyalkanoate Synthase from Aeromonas caviae in Polymerization Reaction

Polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae. FA440 (PhaC(Ac), BAA21815) is one of the most valuable PHA synthase, because of its function to synthesize a. practical bioplastic, poly[(R)-3-hydroxybutytate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)]. However, biochemical activity and active intermediates of PhaC(Ac) have not been clarified until now. In the present study, agency of PhaC(Ac) was cloned and overexpressed by a cell free protein expression. system. Both the polymerization activity and oligomerization behavior of the purified PhaC(Ac) were characterized in order to clarify the active intermediates of PhaC(Ac) based on the hydrodynamic diameters and specific activities of PhaC(Ac). The influences of a substrate, (R)-3-hydroxybutyryl-CoA (3HB-CoA), on the oligomerization of PhaC(Ac) (7.5 mu M), were also investigated, and then the Hill coefficient (n = 2.6 +/- 0.4) and the microscopic dissociation constant (K-m = 77 +/- 5 mu M) were determined. Based on the results, the active intermediate of PhaC(Ac) was concluded to be the dimeric PhaC(Ac) containing 3HB-CoA, as an activator for its dimerization. This information is critical for revealing the relationships between its dimerization and function in PHA synthesis.