Abstract
Summary: Individual polyhydroxyalkanoate synthase molecules from Ralstonia eutropha (PhaC~Re~) were directly visualized on highly oriented pyrolytic graphite (HOPG) by atomic force microscopy (AFM). PhaC~Re~ 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 PhaC~Re~ molecules with (R)‐3‐hydroxybutyryl‐CoA monomers. The reaction product was then observed after the removal of water solution. Several PhaC~Re~ 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 PhaC~Re~ assembly was interpreted as the poly[(R)‐3‐hydroxybutyrate] polymer chain. High resolution AFM suggested that the PhaC~Re~ assembly was composed of 3–4 subunits of PhaC~Re~ molecules. This was further supported by SDS‐PAGE analysis of the cross‐linked PhaC~Re~ enzyme. These results suggest that more than two subunits of PhaC~Re~ are necessary for the in vitro polymerization of PHB molecular chains.
AFM image of PhaC~Re~ molecules after in vitro polymerization reaction for 5 min.
magnified imageAFM image of PhaC~Re~ molecules after in vitro polymerization reaction for 5 min.