Real-time NMR analysis of polyhydroxyalkanoate synthase reaction that synthesizes block copolymer comprising glycolate and 3-hydroxybutyrate.

The sequence-regulating polyhydroxyalkanoate (PHA) synthase PhaC spontaneously synthesizes the homo-random block copolymer, poly[3-hydroxybutyrate (3HB)]-b-poly[glycolate (GL)-ran-3HB]. In this study, a real-time in vitro chasing system was established using a high-resolution 800 MHz nuclear magnetic resonance (NMR) and C-labeled monomers to monitor the polymerization of GL-CoA and 3HB-CoA into this atypical copolymer. Consequently, PhaC initially consumed only 3HB-CoA and subsequently consumed both substrates. The structure of the nascent polymer was analyzed by extracting it with deuterated hexafluoro-isopropanol. In the primary reaction product, a 3HB-3HB dyad was detected, and GL-3HB linkages were subsequently formed. According to these results, the P(3HB) homopolymer segment is synthesized prior to the random copolymer segment. This is the first report of its kind which proposes the application of real-time NMR to a PHA synthase assay, paving the way for elucidating the mechanisms of PHA block copolymerization.