Crystal structure of Ralstonia eutropha polyhydroxyalkanoate synthase C‐terminal domain and reaction mechanisms

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by numerous microorganisms as energy and reducing power storage materials, and have attracted much attention as substitutes for petroleum‐based plastics. Here, we report the first crystal structure of Ralstonia eutropha PHA synthase at 1.8 Å resolution and structure‐based mechanisms for PHA polymerization. RePhaC1 contains two distinct domains, the N‐terminal (RePhaC1ND) and C‐terminal domains (RePhaC1CD), and exists as a dimer. RePhaC1CD catalyzes polymerization via non‐processive ping‐pong mechanism using a Cys‐His‐Asp catalytic triad. Molecular docking simulation of 3‐hydroxybutyryl‐CoA to the active site of RePhaC1CD reveals residues involved in the formation of 3‐hydroxybutyryl‐CoA binding pocket and substrate binding tunnel. Comparative analysis with other polymerases elucidates how different classes of PHA synthases show different substrate specificities. Furthermore, we attempted structure‐based protein engineering and developed a RePhaC1 mutant with enhanced PHA synthase activity. Polyhydroxyalkanoates (PHAs) are bacterial polyesters and have attracted substantial attention as substitutes for the petroleum‐based plastics. In this study, the authors report the crystal structure of C‐terminal domain of PHA synthase from Ralstonia eutropha, the best studied bacterium for PHA production, and the structural basis for the detailed molecular mechanisms of PHA biosynthesis. The structural information with reaction mechanisms will be useful for the rational engineering of PHA synthases to produce designer bioplastics from various monomers more efficiently.