Organization of polyhydroxyalkanoate synthase for in vitro polymerization as revealed by atomic force microscopy.
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.
Kikkawa
Yoshihiro
Y
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
Narike
Masaharu
M
Hiraishi
Tomohiro
T
Kanesato
Masatoshi
M
Sudesh
Kumar
K
Doi
Yoshiharu
Y
Tsuge
Takeharu
T
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
D003432:Cross-Linking Reagents; D011108:Polymers; D012996:Solutions; D014867:Water; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D005976:Glutaral