Organization of polyhydroxyalkanoate synthase for in vitro polymerization as revealed by atomic force microscopy |
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Authors: | Kikkawa Yoshihiro Narike Masaharu Hiraishi Tomohiro Kanesato Masatoshi Sudesh Kumar Doi Yoshiharu Tsuge Takeharu |
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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 |
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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. |
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Keywords: | atomic force microscopy (AFM) enzymes in vitro polymerization PHA synthase polyhydroxyalcanoate (PHA) |
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