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

Eur J Biochem. 1994 Nov 15;226(1):71-80. doi: 10.1111/j.1432-1033.1994.tb20027.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / immunology
  • Acyltransferases / isolation & purification*
  • Acyltransferases / metabolism
  • Acyltransferases / ultrastructure
  • Amino Acid Sequence
  • Antibodies
  • Blotting, Western
  • Chromatium / enzymology*
  • Chromatium / ultrastructure
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Cytoplasmic Granules / enzymology
  • Hydrogen-Ion Concentration
  • Microscopy, Immunoelectron
  • Molecular Sequence Data

Substances

  • Antibodies
  • Acyltransferases
  • poly(3-hydroxyalkanoic acid) synthase