title: Active intermediates of polyhydroxyalkanoate synthase from Aeromonas caviae in polymerization reaction.
abstract: 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-hydroxybutyrate-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, a gene 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 μM) were also investigated, and then the Hill coefficient (n = 2.6 ± 0.4) and the microscopic dissociation constant (K(m) = 77 ± 5 μM) 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.
authors.affiliation: Enzyme Research Team, RIKEN Biomass Engineering Program, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Keiji.numata@riken.jp
authors.forename: Keiji
authors.initials: K
authors.lastname: Numata
authors.forename: Yoko
authors.initials: Y
authors.lastname: Motoda
authors.forename: Satoru
authors.initials: S
authors.lastname: Watanabe
authors.forename: Naoya
authors.initials: N
authors.lastname: Tochio
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Doi
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057585:Aeromonas caviae / Q000201:enzymology* / Q000235:genetics; D055162:Biocatalysis; D002474:Cell-Free System; D003001:Cloning, Molecular; D057075:Enzyme Assays; D058105:Polymerization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase