Plastics and other polymers are used every day, and they are predominantly produced from fossil resources by means of petrochemical refinery process. Conversely, polyesters known as polyhydroxyalkanoates (PHAs) are naturally synthesized by many microorganisms, as discrete granules inside the cells.
PHAs are a family of microbial polyesters, which have been used as biocompatible and biodegradable elastomers and plastics utilized as alternatives to petrochemical counterparts. A number of patents and papers related to gene biochemical studies, cloning and metabolic engineering of PHA biosynthetic machineries, and production of PHAs have been published. A Google search for the term “polyhydroxyalkanoates” provides 223,000 document pages.
PHAs have always been considered to be an astounding example for biological polymer synthesis. Another amazing fact is that PHAs in the range 500 kDa to sometimes as high as 10,000 kDa can be synthesized in vivo using PHA synthase, which is the main polymerizing enzyme for PHA biosynthesis.
Because of this, the determination of crystal structure of PHA synthase has found considerable attention in the past 30 years, but unfortunately without a positive outcome. So, the molecular mechanisms and properties of PHA synthase are still unknown.
On November 30, 2016, a Korean research group led by Professor Kyung-Jin Kim from Kyungpook National University and Distinguished Professor Sang Yup Lee from the Korea Advanced Institute of Science and Technology (KAIST) published two papers back-to-back online in Biotechnology Journal, where they presented an account of the crystal structure of PHA synthase from Ralstonia eutropha, the bacterium best analyzed for PHA synthesis.
The group also presented the structural basis for the detailed molecular mechanisms for the biosynthesis of PHA. In February 2016, the crystal structure was deposited to Protein Data Bank.
Once the research team decoded the crystal structure of the PHA synthase’s catalytic domain along with other structural analysis on whole enzyme and related proteins, they carried out various experiments to throw light on the mechanisms of enzyme reaction, enzyme engineering, validating detailed structures, and N-terminal domain studies, among others.
Through numerous biochemical studies based on crystal structure, the researchers proved that PHA synthase occurs as a dimer and is made of two distinct domains: the C-terminal domain (RePhaC1CD) and the N-terminal domain (RePhaC1ND). The RePhaC1CD utilizes a non-processive ping-pong mechanism with a Cys-His-Asp catalytic triad to catalyze the polymerization reaction.
The clearance between the two catalytic sites of the RePhaC1CD dimer is 33.4 Å, indicating that the polymerization reaction at each site takes place independently. The research also reports the structure-based mechanisms for substrate specificities of different types of PHA syntheses belonging to disparate classes.
The results and information presented in these two papers have been very much awaited not only in the PHA community, but also metabolic engineering, bacteriology/microbiology, and in general biological sciences communities. The structural information on PHA synthase together with reaction mechanisms deciphered will be valuable for understanding the detailed mechanisms of biosynthesizing this important energy/redox storage material, and also for the rational engineering of PHA synthases to produce designer bioplastics from various monomers more efficiently.
Professor Sang Yup Lee, KAIST