Publications

• 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.

  Liebergesell M., Sonomoto K., Madkour M., Mayer F., Steinbuechel A.

  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-hydroxyal ... >> More

  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. << Less

  Eur. J. Biochem. 226:71-80(1994) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• PHA synthase from Chromatium vinosum: cysteine 149 is involved in covalent catalysis.

  Mueh U., Sinskey A.J., Kirby D.P., Lane W.S., Stubbe J.

  Polyhydroxyalkanoate synthase (PHA) from Chromatium vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a approximately 1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in soluti ... >> More

  Polyhydroxyalkanoate synthase (PHA) from Chromatium vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a approximately 1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His)6-tagged construct. The PhaC-(His)6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His)6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His)6 to PhaC-(His)6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ([3H]-sT-CoA). After Sephadex G50 chromatography, the synthase contained approximately 0.25 equiv of the labile label per PhaC. Incubation of [3H]-sT-synthase with HB-CoA resulted in production of [3H]-polymer. Digestion of [3H]-sT-synthase with trypsin and HPLC analysis resulted in isolation of three labeled peptides. Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [3H]-sT while the other two contained, in addition to the [3H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130. << Less

  Biochemistry 38:826-837(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Co-expression of two polyhydroxyalkanoate synthase subunits from Synechocystis sp. PCC 6803 by cell-free synthesis and their specific activity for polymerization of 3-hydroxybutyryl-coenzyme A.

  Numata K., Motoda Y., Watanabe S., Osanai T., Kigawa T.

  Synechocystis sp. PCC 6803 is one of the most studied cyanobacteria for polyhydroxyalkanoate (PHA) synthesis, and its PHA synthase is known to consist of two subunits, namely, PhaC and PhaE. This report is the first to show the specific activity and related biochemical properties of PHA synthase f ... >> More

  Synechocystis sp. PCC 6803 is one of the most studied cyanobacteria for polyhydroxyalkanoate (PHA) synthesis, and its PHA synthase is known to consist of two subunits, namely, PhaC and PhaE. This report is the first to show the specific activity and related biochemical properties of PHA synthase from cyanobacteria. We have cloned and prepared a complex of PhaC and PhaE (PhaCE) from Synechocystis sp. PCC 6803 by the co-expression of PhaC and PhaE using a cell-free synthesis system. The specific activity of PhaCE was comparable to that of the class I PHA synthases, indicating that the low PHA productivity of cyanobacteria is not due to the activity of PHA synthase but may be caused by the other metabolic reactions related to PHA synthesis. The positive Hill coefficient of PhaCE as well as the size exclusion chromatography data indicates that dimeric PhaCE is a major active form that polymerizes 3-hydroxybutyryl-coenzyme A. << Less

  Biochemistry 54:1401-1407(2015) [PubMed] [EuropePMC]