@article {686,
	title = {Identification of a second catalytically active trans-sialidase in Trypanosoma brucei.},
	journal = {Biochem Biophys Res Commun},
	volume = {415},
	year = {2011},
	month = {2011 Nov 18},
	pages = {421-5},
	abstract = {The procyclic stage of Trypanosoma brucei is covered by glycosylphosphatidylinositol (GPI)-anchored surface proteins called procyclins. The procyclin GPI anchor contains a side chain of N-acetyllactosamine repeats terminated by sialic acids. Sialic acid modification is mediated by trans-sialidases expressed on the parasite{\textquoteright}s cell surface. Previous studies suggested the presence of more than one active trans-sialidases, but only one has so far been reported. Here we cloned and examined enzyme activities of four additional trans-sialidase homologs, and show that one of them, Tb927.8.7350, encodes another active trans-sialidase, designated as TbSA C2. In an in vitro assay, TbSA C2 utilized α2-3 sialyllactose as a donor, and produced an α2-3-sialylated product, suggesting that it is an α2-3 trans-sialidase. We suggest that TbSA C2 plays a role in the sialic acid modification of the trypanosome cell surface.},
	keywords = {Amino Sugars, Catalysis, Cell Membrane, Cloning, Molecular, Glycoproteins, Glycosylphosphatidylinositols, Mutation, N-Acetylneuraminic Acid, Neuraminidase, Trypanosoma brucei brucei},
	issn = {1090-2104},
	doi = {10.1016/j.bbrc.2011.10.085},
	author = {Nakatani, Fumiki and Morita, Yasu S and Ashida, Hisashi and Nagamune, Kisaburo and Maeda, Yusuke and Kinoshita, Taroh}
}
@article {862,
	title = {Mechanism of the polymerization reaction initiated and catalyzed by the polyhydroxybutyrate synthase of Ralstonia eutropha.},
	journal = {Biomacromolecules},
	volume = {4},
	year = {2003},
	month = {2003 May-Jun},
	pages = {504-9},
	abstract = {Polyhydroxybutyrate (PHB) synthases (polymerases) catalyze the polymerization of the coenzyme A thioester of 3-hydroxybutyrate to PHB. The Ralstonia eutropha PHB synthase purified from recombinant E. coli cells exists in aqueous solution in both monomeric (single subunit) and homodimeric (two subunits) forms in equilibrium. Several lines of evidence suggest that the homodimer is the active form of the synthase. The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site. The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis, but a second thiol group on the same protein molecule has not yet been identified. It is suggested that cysteines at 319 from each of the two molecules of a homodimer synthase provide two identical thiol groups to jointly form a single catalytic site. To verify this model using the strategy of in vitro reconstitution, heterodimers composed of the wild-type subunit and of the C(319) mutated subunit were constructed and the activities at various ratios of the wild-type subunit to the mutated subunit were measured. The experimental results indicate that the homodimer is the active form of the enzyme, that the heterodimer containing the mutated subunit has no activity, and that a single cysteine is not sufficient for catalysis. Two identical thiol groups from C(319) residues on each subunit of the homodimer are required to form the catalytic site for the initiation and propagation reactions. We further demonstrate that a dimer synthase that has initiated the polymerization reaction (primed synthase) is significantly more stable against dissociation than the unprimed (unreacted) dimer synthase. These two properties explain the nature of lag phenomenon during the in vitro polymerization reaction catalyzed by this enzyme},
	keywords = {Catalysis, Cupriavidus necator, Hydroxybutyrates, Polymers},
	issn = {1525-7797},
	doi = {10.1021/bm020099x},
	author = {Zhang, Shiming and Kolvek, Steve and Lenz, Robert W and Goodwin, Steve}
}
@article {865,
	title = {Kinetic and mechanistic characterization of the polyhydroxybutyrate synthase from Ralstonia eutropha.},
	journal = {Biomacromolecules},
	volume = {1},
	year = {2000},
	month = {2000 Summer},
	pages = {244-51},
	abstract = {Purified Ralstonia eutropha polyhydroxybutyrate (PHB) synthase from recombinant cells can exist as monomer and dimer. The polymerization reaction catalyzed by this enzyme displays a lag phase, which causes difficulties for kinetic and mechanistic characterization of the enzymatic polymerization reaction. In this study, we developed a method to eliminate the lag phase of PHB synthase by physical means, i.e., adding multihydroxyl compounds to the enzyme solution. This method allows us to recognize the nature of the lag phase as a physical rather than a chemical process. With such lag-phase-free-enzyme, the kinetic properties of the enzyme were investigated. The results indicate that 3-hydroxybutyryl-CoA (3HBCoA) is the optimal substrate for the enzyme. A slower catalytic rate and lower binding ability account for a lower reactivity of 3-hydroxyvaleryl-CoA (3HVCoA) compared to that of 3HBCoA. The change of hydroxyl group from the beta to the gamma position causes dramatic decreases in the binding ability of 4-hydroxybutyryl-CoA (4HBCoA). By using a dilution strategy and size exclusion chromatographic technique, the active form of the enzyme was identified to be the dimeric form. The number of catalytic sites in the dimeric form of the enzyme was examined by comparing the molecular weight of polyhydroxybutyrate as a function of substrate-to-enzyme ratio. The results suggest that the dimeric enzyme has only one catalytic site. A revised model of polymerization reaction catalyzed by R. eutropha PHB synthase is described.},
	keywords = {Acyltransferases, Catalysis, Chromatography, Gel, Coenzyme A, Culture Media, Hydrogen-Ion Concentration, Kinetics, Molecular Weight, Proteobacteria},
	issn = {1525-7797},
	author = {Zhang, S and Yasuo, T and Lenz, R W and Goodwin, S}
}
