title: Trapping of intermediates with substrate analog HBOCoA in the polymerizations catalyzed by class III polyhydroxybutyrate (PHB) synthase from Allochromatium vinosum.
abstract: Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the formation of biodegradable PHB polymers that are considered as an ideal alternative to petroleum-based plastics. To provide strong evidence for the preferred mechanistic model involving covalent and noncovalent intermediates, a substrate analog HBOCoA was synthesized chemoenzymatically. Substitution of sulfur in the native substrate HBCoA with an oxygen in HBOCoA enabled detection of (HB)nOCoA (n = 2-6) intermediates when the polymerization was catalyzed by wild-type (wt-)PhaECAv at 5.84 h(-1). This extremely slow rate is due to thermodynamically unfavorable steps that involve the formation of enzyme-bound PHB species (thioesters) from corresponding CoA oxoesters. Synthesized standards (HB)nOCoA (n = 2-3) were found to undergo both reacylation and hydrolysis catalyzed by the synthase. Distribution of the hydrolysis products highlights the importance of the penultimate ester group as previously suggested. Importantly, the reaction between primed synthase [(3)H]-sT-PhaECAv and HBOCoA yielded [(3)H]-sTet-O-CoA at a rate constant faster than 17.4 s(-1), which represents the first example that a substrate analog undergoes PHB chain elongation at a rate close to that of the native substrate (65.0 s(-1)). Therefore, for the first time with a wt-synthase, strong evidence was obtained to support our favored PHB chain elongation model.
authors: Department of Chemistry, Kansas State University, Manhattan, Kansas 66506, United States.
authors: Chao
authors: C
authors: Chen
authors: Department of Chemistry, Kansas State University, Manhattan, Kansas 66506, United States.
authors: Ruikai
authors: R
authors: Cao
authors: Department of Chemistry, Kansas State University, Manhattan, Kansas 66506, United States.
authors: Ruben
authors: R
authors: Shrestha
authors: University of Saint Mary, Leavenworth, Kansas 66048, United States.
authors: Christina
authors: C
authors: Ward
authors: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas 66506, United States.
authors: Benjamin B
authors: BB
authors: Katz
authors: Department of Physics and Astronomy, University of Kansas, Lawrence, Kansas 66045, United States.
authors: Christopher J
authors: CJ
authors: Fischer
authors: Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas 66506, United States.
authors: John M
authors: JM
authors: Tomich
authors: Department of Chemistry, Kansas State University, Manhattan, Kansas 66506, United States.
authors: Ping
authors: P
authors: Li
mesh_terms: D000217:Acyltransferases / Q000378:metabolism*; D002841:Chromatiaceae / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D058105:Polymerization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D003065:Coenzyme A