Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination
Author(s)Buckley, Rachael Marie; Stubbe, JoAnne
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Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the conversion of 3-(R)-hydroxybutyryl CoA (HBCoA) to PHB, which is deposited as granules in the cytoplasm of microorganisms. The class I PhaC from Caulobacter crescentus (PhaC[subscript Cc]) is a highly soluble protein with a turnover number of 75 s[superscript –1] and no lag phase in coenzyme A (CoA) release. Studies with [1-[superscript 14]C]HBCoA and PhaC[subscript Cc] monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography reveal that the rate of elongation is much faster than the rate of initiation. Priming with the artificial primer [[superscript 3]H]sTCoA and monitoring for CoA release reveal a single CoA/PhaC, suggesting that the protein is uniformly loaded and that the elongation process could be studied. Reaction of sT-PhaC[subscript Cc] with [1-[superscript 14]C]HBCoA revealed that priming with sTCoA increased the uniformity of elongation, allowing distinct polymerization species to be observed by SDS–PAGE and autoradiography. However, in the absence of HBCoA, [3H]sT-PhaC unexpectedly generates [3H]sDCoA with a rate constant of 0.017 s[superscript –1]. We propose that the [[superscript 3]H]sDCoA forms via attack of CoA on the oxoester of the [[superscript 3]H]sT-PhaC chain, leaving the synthase attached to a single HB unit. Comparison of the relative rate constants of thiolysis by CoA and elongation by PhaC[subscript Cc], and the size of the PHB polymer generated in vivo, suggests a mechanism for chain termination and reinitiation.
DepartmentMassachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Chemistry
American Chemical Society (ACS)
Rachael M. Buckley, and JoAnne Stubbe. "Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination." Biochemistry 54:12 (2015), pp. 2117-2125. © 2015 American Chemical Society.
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