Mechanistic Insight with HBCH(2)CoA as a Probe to Polyhydroxybutyrate (PHB) Synthases

作者：Zhang Wei; Shrestha Ruben; Buckey Rachael M; Jewell Jamie; Bossmann Stefan H; Stubbe JoAnne^*; Li Ping

来源：ACS Chemical Biology, 2014, 9(8): 1773-1779.

DOI：10.1021/cb5002735

Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1-2 MDa. A substrate analogue HBCH(2)CoA, 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 HI PHB synthases, with K-is of 40 and 14 mu M, respectively. To probe the elongation steps of the polymerization, HBCH(2)CoA was incubated with a synthase acylated with a [H-3]-saturated trimer-CoA ([H-3]-sTCoA). The products of the reaction were shown to be the methylene analogue of [H-3]-sTCoA ([H-3]-sT-CH2-CoA), saturated dimer-([H-3]-sD-CO2H), and trimer-acid ([H-3]-sT-CO2H), distinct from the expected methylene analogue of [H-3]-saturated tetramer-CoA ([H-3]-sTet-CH2-CoA). Detection of [H-3]-sT-CH2-CoA and its slow rate of formation suggest that HBCH(2)CoA may be reporting on the termination and repriming process of the synthases, rather than elongation.

• 出版日期2014-8