Purification of Polyhydroxybutyrate Synthase from Its Native Organism, Ralstonia eutropha: Implications for the Initiation and Elongation of Polymer Formation in Vivo

摘要: Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB (R)-3-hydroxybutyryl-CoA, ultimately resulting in insoluble granules. Previous mechanistic studies R. PhaC, purified Escherichia coli (PhaC(Ec)), demonstrated that polymer elongation rate is much faster than initiation rate. In an effort to identify a factor(s) native organism might prime and increase initiation, N-terminally Strep2-tagged phaC (Strep2-PhaC(Re)) was constructed integrated into genome place wild-type phaC. Strep2-PhaC(Re) expressed by affinity chromatography grown nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) 24 (PHB, 2% weight). Analysis PhaC size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, permeation revealed it unexpectedly copurified with phasin protein, PhaP1, soluble (M(w) = 350 kDa) "high-molecular weight" (HMW) complex monomeric/dimeric (M/D) forms no associated PhaP1 or PHB. Assays monitoring HMW showed lag phase CoA release, contrast M/D PhaC(Re) (and PhaC(Ec)), suggesting fraction has been isolated PHB-primed form. The presence primed nonprimed suggests also vivo. A modified micelle model granule genesis proposed accommodate reported observations.