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 , 39500 and PhaE, M r 40500). 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 400000 and most probably consists of ten subunits. The K m 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.