title: Organization of polyhydroxyalkanoate synthase for in vitro polymerization as revealed by atomic force microscopy.
abstract: Individual polyhydroxyalkanoate synthase molecules from Ralstonia eutropha (PhaCRe) were directly visualized on highly oriented pyrolytic graphite (HOPG) by atomic force microscopy (AFM). PhaCRe molecule was observed as a spherical particle of 2.9 +/- 0.4 nm in height and 28 +/- 4 nm in width. In vitro polymerization reaction on HOPG was carried out for 5 min by reacting the PhaCRe molecules with (R)-3-hydroxybutyryl-CoA monomers. The reaction product was then observed after the removal of water solution. Several PhaCRe molecules associated with each other to form an assembly, which was attached to a fibrillar structure of ca. 0.2-0.3 nm in height. The fibrillar structure that elongated from the PhaCRe assembly was interpreted as the poly[(R)-3-hydroxybutyrate] polymer chain. High resolution AFM suggested that the PhaCRe assembly was composed of 3-4 subunits of PhaCRe molecules. This was further supported by SDS-PAGE analysis of the cross-linked PhaCRe enzyme. These results suggest that more than two subunits of PhaCRe are necessary for the in vitro polymerization of PHB molecular chains.
authors.affiliation: Nanoarchitectonics Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan. y.kikkawa@aist.go.jp
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kikkawa
authors.forename: Masaharu
authors.initials: M
authors.lastname: Narike
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Hiraishi
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Kanesato
authors.forename: Kumar
authors.initials: K
authors.lastname: Sudesh
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Doi
authors.forename: Takeharu
authors.initials: T
authors.lastname: Tsuge
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D003432:Cross-Linking Reagents / Q000737:chemistry; D020118:Cupriavidus necator / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D005976:Glutaral / Q000737:chemistry; D066298:In Vitro Techniques; D018625:Microscopy, Atomic Force; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism*; D012996:Solutions; D014867:Water / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D011108:Polymers; D012996:Solutions; D014867:Water; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D005976:Glutaral