Mechanistic Insight with HBCH2CoA as a Probe to Polyhydroxybutyrate (PHB) Synthases

Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1-2 MDa. A substrate analogue HBCH(2)CoA, in which the S in HBCoA is replaced with a CH2 group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class HI PHB synthases, with K-is of 40 and 14 mu M, respectively. To probe the elongation steps of the polymerization, HBCH(2)CoA was incubated with a synthase acylated with a [H-3]-saturated trimer-CoA ([H-3]-sTCoA). The products of the reaction were shown to be the methylene analogue of [H-3]-sTCoA ([H-3]-sT-CH2-CoA), saturated dimer-([H-3]-sD-CO2H), and trimer-acid ([H-3]-sT-CO2H), distinct from the expected methylene analogue of [H-3]-saturated tetramer-CoA ([H-3]-sTet-CH2-CoA). Detection of [H-3]-sT-CH2-CoA and its slow rate of formation suggest that HBCH(2)CoA may be reporting on the termination and repriming process of the synthases, rather than elongation.