Trapping of Intermediates with Substrate Analog HBOCoA
in the Polymerizations Catalyzed by Class III Polyhydroxybutyrate
(PHB) Synthase from <i>Allochromatium Vinosum</i>
Chao Chen
Ruikai Cao
Ruben Shrestha
Christina Ward
Benjamin
B. Katz
Christopher J. Fischer
John M. Tomich
Ping Li
10.1021/cb5009958.s001
https://acs.figshare.com/articles/journal_contribution/Trapping_of_Intermediates_with_Substrate_Analog_HBOCoA_in_the_Polymerizations_Catalyzed_by_Class_III_Polyhydroxybutyrate_PHB_Synthase_from_i_Allochromatium_Vinosum_i_/2166250
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)<sub><i>n</i></sub>OCoA (<i>n</i> = 2–6) intermediates when the polymerization was catalyzed
by wild-type (<i>wt</i>-)PhaEC<sub>Av</sub> at 5.84 h<sup>–1</sup>. 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)<sub><i>n</i></sub>OCoA (<i>n</i> = 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 [<sup>3</sup>H]-sT-PhaEC<sub>Av</sub> and HBOCoA yielded [<sup>3</sup>H]-sTet-O-CoA at a rate
constant faster than 17.4 s<sup>–1</sup>, 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<sup>–1</sup>). Therefore, for the first time with a <i>wt</i>-synthase,
strong evidence was obtained to support our favored PHB chain elongation
model.
2015-05-15 00:00:00
hydrolysis products
PHB chain elongation model
PHB chain elongation
substrate analog HBOCoA
Class III Polyhydroxybutyrate
Substrate Analog HBOCoA
substrate analog
ester group
noncovalent intermediates
Polymerizations Catalyzed
Synthesized standards
synthase
PHB polymers
CoA oxoesters
Allochromatium VinosumPolyhydroxybutyrate
substrate HBCoA