Class III polyhydroxybutyrate synthase: involvement in chain termination and reinitiation.
Polyhydroxybutyrate (PHB) synthase catalyzes the polymerization of (R)-3-hydroxybutyryl-CoA (CoA = coenzyme A) into high molecular weight PHB. Recombinant wild-type (wt) class III synthase from Allochromatium vinosum (PhaCPhaE(Av)), antibodies to this synthase and to PHB, and [(14)C]hydroxybutyryl-CoA (HB-CoA) have been used to detect oligomeric hydroxybutyrate (HB) units covalently bound to the synthase using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Although a distribution of products is typically observed, short (HB)(n)-bound synthases (designated species I) are most prevalent at low substrate to enzyme (S/E) ratios. Species I is similar to (HB)(n)-PhaC(Av) (n = 3-10 at minimum) recently identified using D302A-PhaCPhaE(Av) (Tian, J., Sinskey, A. J., and Stubbe, J. (2005) Biochemistry 44, 1495-1503). Species I is shown to be an intermediate in the elongation process of PHB synthesis in vitro. The reaction catalyzed by the wt synthase in vitro was further studied under two sets of conditions: at high (70000) and low (<200) S/E ratios. At high S/E ratios, kinetic analysis of the reaction of HB-CoA with the wt synthase monitored using antibodies to PhaCPhaE(Av) and Western blotting revealed the disappearance of PhaC(Av) at early time points and its reappearance as the molecular weight of the PHB approached 1.8 MDa. At low S/E ratios, species I was observed to increase with time after complete consumption of all of the HB-CoA. The results from studies under both sets of conditions suggest that an inherent property of the synthase is chain termination and reinitiation.
Tian
Jiamin
J
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Sinskey
Anthony J
AJ
Stubbe
JoAnne
J
D020155:3-Hydroxybutyric Acid / Q000737:chemistry / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry* / Q000145:classification* / Q000378:metabolism; D001345:Autoradiography; D001426:Bacterial Proteins / Q000737:chemistry* / Q000145:classification* / Q000378:metabolism; D015153:Blotting, Western; D002250:Carbon Radioisotopes / Q000378:metabolism; D002384:Catalysis; D002841:Chromatiaceae / Q000201:enzymology; D020118:Cupriavidus necator / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D011994:Recombinant Proteins / Q000737:chemistry / Q000145:classification / Q000378:metabolism; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D011994:Recombinant Proteins; C030372:3-hydroxybutyryl-coenzyme A; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; C060970:poly-beta-hydroxybutyrate polymerase; D020155:3-Hydroxybutyric Acid