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.
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.
Institut für Mikrobiologie, Georg-August-Universität zu Göttingen, Germany.
M
M
Liebergesell
K
K
Sonomoto
M
M
Madkour
F
F
Mayer
A
A
Steinbüchel
D000217:Acyltransferases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000906:Antibodies; D015153:Blotting, Western; D002844:Chromatium / Q000201:enzymology* / Q000648:ultrastructure; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003594:Cytoplasmic Granules / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data
D000906:Antibodies; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase