Purification and Characterization of the Poly(Hydroxyalkanoic Acid) Synthase from Chromatium vinosum and Localization of the Enzyme at the Surface of Poly(Hydroxyalkanoic Acid) Granules

Abstract

A recombinant strain of Escherichia coli, which overexpressed phaC and phaE from Chromatium vinosum, was used to isolate poly(3-hydroxyalkanoic acid) synthase. The isolation was performed by a two-step procedure including chromatography on DEAE-Sephacel and Procion Blue H-ERD. The poly(3-hydroxyalkanoic acid) synthase consisted of two different kinds of subunit (PhaC, M(r) 39,500 and PhaE, M(r) 40.500). PhaC was separated from the poly(3-hydroxyalkanoic acid) synthase complex by chromatography on phenyl-Sepharose: PhaE was enriched by solubilization of protein inclusion bodies. The stoichiometry of PhaC and PhaE in the enzyme complex was not determined. The poly(3-hydroxyalkanoic acid) synthase (PhaEC) exhibited a native relative molecular mass of M(r) 400,000 and most probably consists of ten subunits. The Km value of the enzyme for D(-)-3-hydroxybutyryl-CoA was 0.063 mM. The enzyme synthesized poly(3-hydroxybutyric acid) in vitro from D(-)-3-hydroxybutyryl-CoA or, together with propionyl-CoA transferase in a coupled enzyme reaction, synthesized the same product from acetyl-CoA plus D(-)-3-hydroxybutyric acid. Antibodies were raised against both subunits of the poly(3-hydroxyalkanoic acid) synthase. By immunoelectron microscopy, the poly(3-hydroxyalkanoic acid) synthase was localized within the cytoplasm in cells of C. vinosum grown under non-storage conditions. In cells grown under poly(3-hydroxybutyric acid) storage conditions, the enzyme was observed to be located at the surface of the poly(3-hydroxybutyric acid) granules. Immunoblots with anti-PhaC, anti-PhaE IgG and crude extract proteins indicated that poly(3-hydroxyalkanoic acid) synthases with partial sequence similarities are widespread among purple sulphur bacteria.

PHA Synthase from Chromatium vinosum: Cysteine 149 Is Involved in Covalent Catalysis

“…Studies of the Steinbüchel group laid the groundwork for a detailed mechanistic study on the class III PHA synthases (11,36,29). The synthase from C. Vinosum differs markedly in structure from the R. eutropha class I synthase (37,10), while maintaining a similar substrate specificity.…”

“…Previous studies, using rocket immunoelectrophoresis, reported a ratio of PhaE to PhaC of 4:1 to 1.5:1 (36). We have reinvestigated the subunit stoichiometry by several methods.…”

“…Studies by Steinbüchel reported cloning, sequencing, and expression of the C. Vinosum synthase (11,36,29). They purified PHA synthase to homogeneity and reported a specific activity of 60 units/mg.…”

“…They demonstrated that it was composed of two subunits, designated PhaE and PhaC (36). PhaC from C. Vinosum, while possessing low sequence identity with class I synthases (6%), possesses all of the conserved residues.…”

Optimizing acidic methanolysis of poly(3‐hydroxyalkanoates) in gas chromatography analysis

“…[18] The samples (pure PHAs or wetted cell) were dissolved and acidified in 2 ml of methanol with four concentrations [1,3,5 or 15% (v/v)] of H 2 SO 4 , and 2 ml of chloroform in a screw-capped test tube. The solution was then kept at 100…”