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. RePhaC1CDcatalyzes 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 RePhaC1CDreveals 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.
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea.
Jieun
J
Kim
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea.
Yeo-Jin
YJ
Kim
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, and Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
So Young
SY
Choi
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, and Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
Sang Yup
SY
Lee
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea.
Kyung-Jin
KJ
Kim
D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D020118:Cupriavidus necator / Q000201:enzymology*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D016296:Mutagenesis; D000072417:Protein Domains; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; C030372:3-hydroxybutyryl-coenzyme A; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase
Crystal structure; Enzyme mechanism; PHA synthase; Polyhydroxyalkanoates; Ralstonia eutropha