title: Mechanistic insight with HBCH2CoA 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 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 [(3)H]-saturated trimer-CoA ([(3)H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [(3)H]-sTCoA ([(3)H]-sT-CH2-CoA), saturated dimer-([(3)H]-sD-CO2H), and trimer-acid ([(3)H]-sT-CO2H), distinct from the expected methylene analogue of [(3)H]-saturated tetramer-CoA ([(3)H]-sTet-CH2-CoA). Detection of [(3)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.
authors.affiliation: Department of Chemistry, Kansas State University , Manhattan, Kansas 66506, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Ruben
authors.initials: R
authors.lastname: Shrestha
authors.forename: Rachael M
authors.initials: RM
authors.lastname: Buckley
authors.forename: Jamie
authors.initials: J
authors.lastname: Jewell
authors.forename: Stefan H
authors.initials: SH
authors.lastname: Bossmann
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
authors.forename: Ping
authors.initials: P
authors.lastname: Li
mesh_terms: D000215:Acylation; D007700:Kinetics; D008025:Ligases / Q000737:chemistry*; D015335:Molecular Probes; D058105:Polymerization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D015335:Molecular Probes; D008025:Ligases