Class III polyhydroxybutyrate synthase: involvement in chain termination and reinitiation

Area of Science:

• Biochemistry
• Polymer Science
• Enzymology

Background:

• Polyhydroxybutyrate (PHB) synthase catalyzes the polymerization of (R)-3-hydroxybutyryl-CoA into PHB.
• Understanding the mechanism of PHB synthesis, including intermediate species, is crucial for metabolic engineering and biopolymer production.

Purpose of the Study:

• To investigate the in vitro reaction mechanism of wild-type (wt) class III PHB synthase from Allochromatium vinosum (PhaCPhaE(Av)).
• To identify and characterize intermediate species in PHB polymerization.
• To elucidate the role of substrate-to-enzyme ratios on PHB synthesis kinetics and product distribution.

Main Methods:

• Utilized recombinant wt PhaCPhaE(Av), specific antibodies, and radiolabeled [(14)C]hydroxybutyryl-CoA (HB-CoA).
• Employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to detect HB units bound to the synthase.
• Performed kinetic analysis and Western blotting under varying substrate-to-enzyme (S/E) ratios (high and low).

Main Results:

• Short oligomeric hydroxybutyrate units bound to the synthase (Species I) were identified as intermediates, particularly prevalent at low S/E ratios.
• At high S/E ratios, synthase disappearance and reappearance were observed as PHB molecular weight increased, suggesting a dynamic process.
• At low S/E ratios, Species I accumulated over time even after substrate depletion, indicating its role in chain elongation and termination/reinitiation.

Conclusions:

• The PHB synthase exhibits an inherent property of chain termination and reinitiation during polymerization.
• Species I represents a key intermediate in the PHB synthesis pathway, linking substrate availability to enzyme activity.
• The findings provide insights into the catalytic mechanism of PHB synthase and its regulation by substrate concentration.