title: Class I and III polyhydroxyalkanoate synthases from Ralstonia eutropha and Allochromatium vinosum: characterization and substrate specificity studies.
abstract: Class I and III polyhydroxyalkanoate (PHA) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to polyhydroxybutyrate. The Class I PHA synthase from Ralstonia eutropha has been purified by numerous labs with reported specific activities that vary between 1 and 160 U/mg. An N-terminal (His)6-PHA synthase was constructed and purified with specific activity of 40 U/mg. The variable activity is shown to be related to the protein's propensity to aggregate and not to incomplete post-translational modification by coenzyme A and a phosphopantetheinyl transferase. The substrate specificities of this enzyme and the Class III PHA synthase from Allochromatium vinosum have been determined with nine analogs of varied chain length and branching, OH group position within the chain, and thioesters. The results suggest that in vitro, both PHA synthases are very specific and provide further support for their active site structural similarities. In vitro results differ from studies in vivo.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
authors.forename: W
authors.initials: W
authors.lastname: Yuan
authors.forename: Y
authors.initials: Y
authors.lastname: Jia
authors.forename: J
authors.initials: J
authors.lastname: Tian
authors.forename: K D
authors.initials: KD
authors.lastname: Snell
authors.forename: U
authors.initials: U
authors.lastname: Müh
authors.forename: A J
authors.initials: AJ
authors.lastname: Sinskey
authors.forename: R H
authors.initials: RH
authors.lastname: Lambalot
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
authors.forename: J
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D000217:Acyltransferases / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020562:Betaproteobacteria / Q000201:enzymology*; D001665:Binding Sites; D003065:Coenzyme A / Q000378:metabolism; D020563:Gammaproteobacteria / Q000201:enzymology*; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011993:Recombinant Fusion Proteins; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D003065:Coenzyme A