title: In vitro polymerization and copolymerization of 3-hydroxypropionyl-CoA with the PHB synthase from Ralstonia eutropha.
abstract: The poly(3-hydroxybutyrate) (PHB) synthase of Ralstonia eutropha, which was produced by a recombinant strain of Escherichia coli and purified in one step with a methyl-HIC column to a purity of more than 90%, was used to polymerize 3-hydroxypropionyl-CoA (3HPCoA) and to copolymerize 3HPCoA with 3-hydroxybutyryl-CoA (3HBCoA). A Km of 189 microM and a kcat of 10 s-1 were determined for the activity of the enzyme in the polymerization reaction of 3HPCoA based on the assumption that the dimer form of PHB synthase was the active form. Free coenzyme A was found to be a very effective competitive inhibitor for the polymerization of 3HPCoA with a Ki of 85 microM. The maximum degree of conversion of 3HPCoA to polymer was less than 40%. In the simultaneous copolymerization reactions of these two monomers, both the turnover number for the copolymerization reaction and the maximum degree of conversion of 3HPCoA and 3HBCoA to copolymers increased with an increase in the amount of 3HBCoA in the monomer mixture. However, the maximum conversion of 3HPCoA to copolymer was always less than 35%, regardless of the ratio of 3HPCoA to 3HBCoA. Block copolymers were obtained by the sequential copolymerization of the two monomers and these copolymers had a much narrower molecular weight distribution than those obtained by the simultaneous copolymerization for the same molar ratio of 3HPCoA to 3HBCoA.
authors.affiliation: Polymer Science and Engineering Department, Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Song
authors.forename: S
authors.initials: S
authors.lastname: Zhang
authors.forename: R W
authors.initials: RW
authors.lastname: Lenz
authors.forename: S
authors.initials: S
authors.lastname: Goodwin
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000302:isolation & purification; D003065:Coenzyme A / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight
chemical_list: D000217:Acyltransferases; C060970:poly-beta-hydroxybutyrate polymerase; D003065:Coenzyme A