Mechanistic Insight with HBCH[subscript 2]CoA as a Probe to Polyhydroxybutyrate (PHB) Synthases
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Stubbe_Mechanistic insight.pdf
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Author(s)
Zhang, Wei
Shrestha, Ruben
Buckley, Rachael M.
Jewell, Jamie
Bossmann, Stefan H.
Stubbe, JoAnne
Li, Ping
Stubbe, JoAnne
Date Issued
June 2014
Journal
ACS Chemical Biology
Publisher
American Chemical Society (ACS)
Citation
Zhang, Wei, Ruben Shrestha, Rachael M. Buckley, Jamie Jewell, Stefan H. Bossmann, JoAnne Stubbe, and Ping Li. “Mechanistic Insight with HBCH[subscript 2]CoA as a Probe to Polyhydroxybutyrate (PHB) Synthases.” ACS Chemical Biology 9, no. 8 (August 15, 2014): 1773–1779. © 2014 American Chemical Society
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Final published version
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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1021/cb5002735
