Chemistry with an artificial primer of polyhydroxybutyrate synthase suggests a mechanism for chain termination.
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. Comparison of the relative rate constants of thiolysis by CoA and elongation by PhaC(Cc), and the size of the PHB polymer generated in vivo, suggests a mechanism for chain termination and reinitiation.
†Department of Chemistry and ‡Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
Rachael M
RM
Buckley
†Department of Chemistry and ‡Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
JoAnne
J
Stubbe
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D016935:Caulobacter crescentus / Q000201:enzymology / Q000235:genetics; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000094122:Polyhydroxybutyrates
D000214:Acyl Coenzyme A; D000217:Acyltransferases; D001426:Bacterial Proteins; D006885:Hydroxybutyrates; D011091:Polyesters; D011994:Recombinant Proteins; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C030372:3-hydroxybutyryl-coenzyme A; C060970:poly-beta-hydroxybutyrate polymerase