Chemistry with an artificial primer of polyhydroxybutyrate synthase suggests a mechanism for chain termination

Biochemistry
Rachael M Buckley, J Stubbe

Abstract

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(Cc)) is a highly soluble protein with a turnover number of 75 s(-1) and no lag phase in coenzyme A (CoA) release. Studies with [1-(14)C]HBCoA and PhaC(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 [(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(Cc) with [1-(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, [(3)H]sT-PhaC unexpectedly generates [(3)H]sDCoA with a rate constant of 0.017 s(-1). We propose that the [(3)H]sDCoA forms via attack of CoA on the oxoester of the [(3)H]sT-PhaC chain, leaving the synthase attached to a single HB unit. Co...Continue Reading

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Citations

Oct 16, 2016·The Journal of Biological Chemistry·Elizabeth C WittenbornCatherine L Drennan

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Methods Mentioned

BETA
acylation
electrophoresis

Software Mentioned

ImageQuant

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