[1S03e] 06. Enzyme Engineering/Enzymology - Part 3

6:15 PM - 6:30 PM JST(9:15 AM - 9:30 AM UTC)

[1ENZ-20] Structural Insights into PHA synthase for Biodegradable Plastic Production

○MIN FEY CHEK¹, Sun-Yong Kim¹, Tomoyuki Mori¹, Keiji Matsumoto², Shunsuke Sato², Toshio Hakoshima¹ (1. Nara Institute of Science and Technology (Japan), 2. KANEKA CORPORATION (Japan))

Keywords:

Biodegradable Plastic,Polyhydroxyalkanoate,Crystal structure,Full-length PhaC,Egress,Dimer

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that have emerged as promising alternatives to petrochemical plastics, a major source of environmental microplastic pollution. PHA synthase (PhaC) is the key enzyme that polymerizes hydroxyacyl-CoA substrates into high-molecular-weight polymers and determines the properties of the PHA produced. Despite its industrial importance, the detailed catalytic mechanism of PhaC remains poorly understood, largely due to the lack of structural information. Therefore, this study aims to elucidate the structural basis of PhaC catalysis using a full-length enzyme from Aeromonas caviae, a highly efficient and industrially relevant PhaC. We determined the crystal structures of full-length PhaC from Aeromonas caviae using X-ray crystallography. The protein was expressed, purified, and crystallized under optimized conditions, followed by structure determination and refinement. Structural analyses focused on domain organization, dimerization interfaces, and internal cavities. Comparative analyses with previously reported structures were conducted to identify key features involved in PHA biosynthesis, particularly substrate entry and product egress pathways.We determined the full-length crystal structures of Aeromonas caviae PhaC in three states: apo (PDB: 9KNK), glycerol-bound (9KNJ), and triethylene glycol-bound (9KNL). These structures reveal a novel product egress tunnel connecting the catalytic pocket to the solvent surface, as well as an N-terminal domain that mediates dimerization. Structural comparison with previously reported free (5XAV) and CoA-bound (6K3C) forms of the C-terminal domain of Chromobacterium sp. USM2 PhaC suggests that the substrate entry site is located near the dynamic LID region.These findings suggest that PhaC catalysis is a dynamic and complex process involving substrate entry, catalytic polymerization with concomitant release of coenzyme A, and subsequent elongation of the polymer chain followed by its exit through the product egress tunnel. Furthermore, PhaC likely undergoes conformational changes to enable substrate access to the catalytic pocket. In addition, previously identified beneficial mutations are located along the proposed product egress pathway, highlighting its functional importance in catalysis.In conclusion, this study presents the first full-length crystal structures of Aeromonas caviae PhaC and identifies key structural elements underlying its catalytic mechanism. The discovery of an N-terminal dimerization domain and a putative product egress tunnel provides fundamental insights into PHA biosynthesis. These findings establish a structural basis for rational enzyme engineering and contribute to the development of more efficient biocatalysts for sustainable biodegradable plastic production.