Mechanistic Insight with HBCH[subscript 2]CoA 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 HBCH2CoA, 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 III PHB synthases, with Kis of 40 and 14 μM, respectively. To probe the elongation steps of the polymerization, HBCH2CoA was incubated with a synthase acylated with a [H]-saturated trimer-CoA ([H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [H]-sTCoA ([H]-sT-CH2-CoA), saturated dimer-([H]-sD-CO2H), and trimer-acid ([ H]-sT-CO2H), distinct from the expected methylene analogue of [H]-saturated tetramer-CoA ([H]-sTet-CH2-CoA). Detection of [ H]-sT-CH2-CoA and its slow rate of formation suggest that HBCH2CoA may be reporting on the termination and repriming process of the synthases, rather than elongation. P (PHAs) are carbon and energy storage polymers synthesized by a variety of bacteria when they find themselves limited in an essential nutrient such as nitrogen or phosphorus but with an abundant carbon source available. Under conditions of maximum accumulation, PHAs can constitute up to 90% of the cell dry weight. When the limiting nutrient is restored, the bacteria degrade the PHAs to release energy and monomers for other biological processes. PHAs are of general interest as they can provide a biodegradable alternative to environmentally unfriendly, petroleum-based plastics. PHA synthases (PhaCs) catalyze the polymerization of 3(R)-hydroxyalkanoate coenzyme A (CoA) to form PHA. They have been divided into four classes based on their substrate specificity, subunit composition, and molecular weight. Class I and III PhaCs utilize short-chain-length monomers such as 3(R)-hydroxybutyrate (HB) coenzyme A (HBCoA) to generate polyhydroxybutyrate (PHB) polymers. The synthase from Ralstonia eutropha (PhaCRe) is the prototypical class I enzyme and is a dimer, composed of two 65 kDa subunits. The synthase from Allochromatium vinosum (PhaCAv) is the prototypical class III synthase and is a tetramer of two ∼40 kDa subunits: PhaC (the synthase) and PhaE (a protein of unknown function that is essential for activity). Studying the mechanism of polymerization (the priming and initiation, elongation, and termination) has been challenging because the elongation process is much faster than the initiation process. Since PhaCs are largely responsible for determining the molecular weight and polydispersity of the PHA, understanding the PhaC mechanism can contribute to the ultimate goal of producing PHA products in an economically competitive