title: Purification of polyhydroxybutyrate synthase from its native organism, Ralstonia eutropha: implications for the initiation and elongation of polymer formation in vivo.
abstract: Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB from (R)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of R. eutropha PhaC, purified from Escherichia coli (PhaC(Ec)), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged phaC (Strep2-PhaC(Re)) was constructed and integrated into the R. eutropha genome in place of wild-type phaC. Strep2-PhaC(Re) was expressed and purified by affinity chromatography from R. eutropha grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) and 24 h (PHB, 2% cell dry weight). Analysis of the purified PhaC by size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography revealed that it unexpectedly copurified with the phasin protein, PhaP1, and with soluble PHB (M(w) = 350 kDa) in a "high-molecular weight" (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays for monitoring the formation of PHB in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaC(Re) (and PhaC(Ec)), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and nonprimed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Mimi
authors.initials: M
authors.lastname: Cho
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Brigham
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Sinskey
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D000214:Acyl Coenzyme A / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D020118:Cupriavidus necator / Q000201:enzymology* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006006:Glycogen Synthase / Q000302:isolation & purification* / Q000378:metabolism; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D006885:Hydroxybutyrates; D046911:Macromolecular Substances; C030372:3-hydroxybutyryl-coenzyme A; D006006:Glycogen Synthase