Publications

• Natural and engineered polyhydroxyalkanoate (PHA) synthase: key enzyme in biopolyester production.

  Zou H., Shi M., Zhang T., Li L., Li L., Xian M.

  With the finite supply of petroleum and increasing concern with environmental issues associated with their harvest and processing, the development of more eco-friendly, sustainable alternative biopolymers that can effectively fill the role of petro-polymers has become a major focus. Polyhydroxyalk ... >> More

  With the finite supply of petroleum and increasing concern with environmental issues associated with their harvest and processing, the development of more eco-friendly, sustainable alternative biopolymers that can effectively fill the role of petro-polymers has become a major focus. Polyhydroxyalkanoate (PHA) can be naturally produced by many species of bacteria and the PHA synthase is believed to be key enzyme in this natural pathway. Natural PHA synthases are diverse and can affect the properties of the produced PHAs, such as monomer composition, molecular weights, and material properties. Moreover, recent studies have led to major advances in the searching of PHA synthases that display specific properties, as well as engineering efforts that offer more efficient PHA synthases, increased PHA compound production, or even novel biopolyesters which cannot be naturally produced. In this article, we review the updated information of natural PHA synthases and their engineering strategies for improved performance in polyester production. We also speculate future trends on the development of robust PHA synthases and their application in biopolyester production. << Less

  Appl Microbiol Biotechnol 101:7417-7426(2017) [PubMed] [EuropePMC]

• 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.

• Biosynthesis and composition of bacterial poly(hydroxyalkanoates).

  Anderson A.J., Haywood G.W., Dawes E.A.

  It is well established that Alcaligenes eutrophus can accumulate a copolymer containing 3-hydroxybutyrate and 3-hydroxyvalerate, but longer 3-hydroxyacid monomers have not been reported to occur in this organism. The properties of the enzymes of poly(hydroxyalkanoate) (PHA) biosynthesis are discus ... >> More

  It is well established that Alcaligenes eutrophus can accumulate a copolymer containing 3-hydroxybutyrate and 3-hydroxyvalerate, but longer 3-hydroxyacid monomers have not been reported to occur in this organism. The properties of the enzymes of poly(hydroxyalkanoate) (PHA) biosynthesis are discussed and it is proposed that the substrate specificity of the polymerizing enzyme restricts the range of monomer units incorporated into PHA. Various other bacteria produce similar copolymers from propionic acid and/or valeric acid. A number of Pseudomonas species accumulate PHAs containing longer-chain monomer units from linear alkanoic acids, alkanes and alcohols. << Less

  Int J Biol Macromol 12:102-105(1990) [PubMed] [EuropePMC]

• Recovery of active medium-chain-length-poly-3-hydroxyalkanoate polymerase from inactive inclusion bodies using ion-exchange resin.

  Ren Q., De Roo G, Kessler B., Witholt B.

  A novel process for the purification of active medium-chain-length-polyhydroxyalkanoate (mcl-PHA) polymerase was developed. This process is based on solubilization and activation of inactive polymerase inclusion bodies by incubation with ion-exchange resin. The mcl-PHA polymerase 1 from Pseudomona ... >> More

  A novel process for the purification of active medium-chain-length-polyhydroxyalkanoate (mcl-PHA) polymerase was developed. This process is based on solubilization and activation of inactive polymerase inclusion bodies by incubation with ion-exchange resin. The mcl-PHA polymerase 1 from Pseudomonas oleovorans was overproduced from the Palk promoter. Most of the polymerase produced was sequestered in the cytoplasm as an inactive form in insoluble aggregates. By incubating the protein aggregates with S-Sepharose ion-exchange resin in the presence of dithiothreitol and glycerol, the mcl-PHA polymerase could be extracted in an active and soluble form with a final yield of about 5.2 mg/g of cell dry weight. The solubilized polymerase was able to catalyse the in vitro synthesis of mcl-PHA without any additional cell components, suggesting its potential application for production of biopolymer. The procedure used here may be of general value in solubilizing and activating purified inactive labile enzymes. << Less

  Biochem J 349:599-604(2000) [PubMed] [EuropePMC]

• Comparison of medium-chain-length polyhydroxyalkanoates synthases from Pseudomonas mendocina NK-01 with the same substrate specificity.

  Guo W., Duan J., Geng W., Feng J., Wang S., Song C.

  The medium-chain-length polyhydroxyalkanoate (PHAMCL) synthase genes phaC1 and phaC2 of Pseudomonas mendocina NK-01 were cloned and inserted into expression plasmid pBBR1MCS-2 to form pBBR1MCS-C1 and pBBR1MCS-C2 which were expressed respectively in the PHAMCL-negative strain P. mendocina C7 whose ... >> More

  The medium-chain-length polyhydroxyalkanoate (PHAMCL) synthase genes phaC1 and phaC2 of Pseudomonas mendocina NK-01 were cloned and inserted into expression plasmid pBBR1MCS-2 to form pBBR1MCS-C1 and pBBR1MCS-C2 which were expressed respectively in the PHAMCL-negative strain P. mendocina C7 whose PHAMCL synthesis operon was defined knock out. P. mendocina C7 derivatives P. mendocina C7C1 and C7C2 carrying pBBR1MCS-C1 and pBBR1MCS-C2 respectively were constructed. Fermentation and gel permeation chromatography (GPC) revealed that P. mendocina C7C1 had higher PHAMCL production rate but its PHAMCL had lower molecular weight than that of P. mendocina C7C2. Gas chromatograph/mass spectrometry (GC/MS) analysis revealed that the two PHAMCL had similarity in monomer composition with 3HD as the favorite monomer i.e. PhaC1 and PhaC2 had the same substrate specificity. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and X-ray diffraction (XRD) also revealed that the two PHAMCL had the same physical properties. P. mendocina NK-01was the first reported strain whose PHAMCL synthases PhaC1 and PhaC2 had the same substrate specificity. << Less

  Microbiol Res 168:231-237(2013) [PubMed] [EuropePMC]

• 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.

• Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism.

  Jia Y., Yuan W., Wodzinska J., Park C., Sinskey A.J., Stubbe J.

  The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutropha and Chromatium vinosum, respectively, catalyze the polymerization of beta-hydroxybutyryl-coenzyme A (HBCoA) to generate PHB. These synthases have different molecular weights, subunit composition, and kinetic properties ... >> More

  The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutropha and Chromatium vinosum, respectively, catalyze the polymerization of beta-hydroxybutyryl-coenzyme A (HBCoA) to generate PHB. These synthases have different molecular weights, subunit composition, and kinetic properties. Recent studies with the C. vinosum synthase suggested that it is structurally homologous to bacterial lipases and allowed identification of active site residues important for catalysis [Jia, Y., Kappock, T. J., Frick, T., Sinskey, A. J., and Stubbe, J. (2000) Biochemistry 39, 3927-3936]. Sequence alignments between the Class I and III synthases revealed similar residues in the R. eutropha synthase. Site-directed mutants of these residues were prepared and examined using HBCoA and a terminally saturated trimer of HBCoA (sT-CoA) as probes. These studies reveal that the R. eutropha synthase possesses an essential catalytic dyad (C319-H508) in which the C319 is involved in covalent catalysis. A conserved Asp, D480, was shown not to be required for acylation of C319 by sT-CoA and is proposed to function as a general base catalyst to activate the hydroxyl of HBCoA for ester formation. Studies of the [(3)H]sT-CoA with wild-type and mutant synthases reveal that 0.5 equiv of radiolabel is covalently bound per monomer of synthase, suggesting that a dimeric form of the enzyme is involved in elongation. These studies, in conjunction with search algorithms for secondary structure, suggest that the Class I and III synthases are mechanistically similar and structurally homologous, despite their physical and kinetic differences. << Less

  Biochemistry 40:1011-1019(2001) [PubMed] [EuropePMC]