Mechanistic Insight with HBCH[subscript 2]CoA as a Probe to Polyhydroxybutyrate (PHB) Synthases

Abstract

Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1–2 MDa. A substrate analogue HBCH[subscript 2]CoA, in which the S in HBCoA is replaced with a CH[subscript 2] 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 III PHB synthases, with K[subscript is] of 40 and 14 μM, respectively. To probe the elongation steps of the polymerization, HBCH[subscript 2]CoA was incubated with a synthase acylated with a [[superscript 3]H]-saturated trimer-CoA ([[superscript 3]H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [[superscript 3]H]-sTCoA ([[superscript 3]H]-sT-CH[subscript 2]-CoA), saturated dimer-([[superscript 3]H]-sD-CO[subscript 2]H), and trimer-acid ([[superscript 3]H]-sT-CO[subscript 2]H), distinct from the expected methylene analogue of [[superscript 3]H]-saturated tetramer-CoA ([[superscript 3]H]-sTet-CH[subscript 2]-CoA). Detection of [[superscript 3]H]-sT-CH[subscript 2]-CoA and its slow rate of formation suggest that HBCH[subscript 2]CoA may be reporting on the termination and repriming process of the synthases, rather than elongation.