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Active Intermediates of Polyhydroxyalkanoate Synthase from Aeromonas caviae in Polymerization Reaction

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journal contribution
posted on 20.02.2016, 06:38 by Keiji Numata, Yoko Motoda, Satoru Watanabe, Naoya Tochio, Takanori Kigawa, Yoshiharu Doi
Polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae FA440 (PhaCAc, BAA21815) is one of the most valuable PHA synthase, because of its function to synthesize a practical bioplastic, poly­[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P­(3HB-co-3HHx)]. However, biochemical activity and active intermediates of PhaCAc have not been clarified until now. In the present study, a gene of PhaCAc was cloned and overexpressed by a cell-free protein expression system. Both the polymerization activity and oligomerization behavior of the purified PhaCAc were characterized in order to clarify the active intermediates of PhaCAc based on the hydrodynamic diameters and specific activities of PhaCAc. The influences of a substrate, (R)-3-hydroxybutyryl-CoA (3HB-CoA), on the oligomerization of PhaCAc (7.5 μM) were also investigated, and then the Hill coefficient (n = 2.6 ± 0.4) and the microscopic dissociation constant (Km = 77 ± 5 μM) were determined. Based on the results, the active intermediate of PhaCAc was concluded to be the dimeric PhaCAc 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.