Comparative study of the relationship between monomer structure and reactivity for two polyhydroxyalkanoate synthases

Area of Science:

• Biochemistry and Molecular Biology
• Polymer Science
• Microbial Biotechnology

Background:

• Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by microorganisms.
• PHA synthases are enzymes responsible for catalyzing PHA polymerization.
• Understanding PHA synthase activity is crucial for developing novel biomaterials.

Purpose of the Study:

• To investigate the substrate specificity and activity of two short-chain PHA synthases: Ralstonia eutropha PHA synthase (type I) and Ectothiorhodospira shaposhnikovii PHA synthase (type III).
• To explore the influence of monomer structure, including side-chain length, hydrophobicity, and hydroxyl group position/configuration, on PHA synthase activity.
• To assess the potential of an in vitro system for synthesizing novel PHA polymers.

Main Methods:

• Utilized organically synthesized hydroxyalkanoate coenzyme A thioesters as substrates.
• Assayed the enzymatic activity of purified R. eutropha PHA synthase and E. shaposhnikovii PHA synthase.
• Systematically varied monomer side-chain length, hydrophobicity, and hydroxyl group characteristics.

Main Results:

• Both PHA synthases exhibited similar activity profiles across various tested monomers.
• 3-Hydroxybutyryl CoA was identified as the most efficient substrate for both enzymes.
• Monomer reactivity was significantly affected by side-chain length (shorter chains reduced activity more severely), hydrophobicity, hydroxyl group configuration ([S] configuration was not recognized), and hydroxyl group position (alpha-carbon substitution drastically reduced activity compared to gamma-carbon).

Conclusions:

• PHA synthase activity is highly dependent on specific structural features of the hydroxyalkanoate monomer.
• The in vitro enzymatic system allows for the preparation of novel PHA polymers not accessible through microbial fermentation due to metabolic constraints.
• This research provides insights into enzyme-substrate interactions and opens avenues for tailored PHA production.