@article {866,
	title = {Comparative study of the relationship between monomer structure and reactivity for two polyhydroxyalkanoate synthases.},
	journal = {Appl Microbiol Biotechnol},
	volume = {56},
	year = {2001},
	month = {2001 Jul},
	pages = {131-6},
	abstract = {Using organically synthesized hydroxyalkanoate coenzyme A thioesters, the activities of two short-chain polyhydroxalkanoate (PHA) synthases were investigated--Ralstonia eutropha PHA synthase (a type I PHA synthase) and Ectothiorhodospira shaposhnikovii PHA synthase (a type III synthase). The results indicate that the two synthases have similar activities towards most of the monomers tested. 3-Hydroxybutyryl CoA was found to be the most efficient substrate for both synthases. Changes in the side-chain length of the monomers affect monomer reactivity, with shortening of the side-chain length having the more severe effect. Hydrophobicity in the side chain appears to play an important role in the catalytic reaction. The configuration and the position of the hydroxyl group also affect the reactivity of a monomer. Monomers with the [S] configuration can not be recognized by either synthase. Moving the hydroxyl group from the beta carbon to the alpha carbon has a much more severe effect on the reactivity of the monomer than moving the hydroxyl group to the gamma carbon. The results demonstrate that the in vitro system can be used to prepare entirely novel polymers that may not be obtainable from living cells because of metabolic restrictions.},
	keywords = {Acyl Coenzyme A, Acyltransferases, Structure-Activity Relationship, Substrate Specificity},
	issn = {0175-7598},
	author = {Zhang, S and Kamachi, M and Takagi, Y and Lenz, R W and Goodwin, S}
}
@article {868,
	title = {Characterization, seasonal occurrence, and diel fluctuation of poly(hydroxyalkanoate) in photosynthetic microbial mats.},
	journal = {Appl Environ Microbiol},
	volume = {66},
	year = {2000},
	month = {2000 Oct},
	pages = {4279-91},
	abstract = {In situ poly(hydroxyalkanoate) (PHA) levels and repeating-unit compositions were examined in stratified photosynthetic microbial mats from Great Sippewissett Salt Marsh, Mass., and Ebro Delta, Spain. Unlike what has been observed in pure cultures of phototrophic bacteria, the prevalence of hydroxyvalerate (HV) repeating units relative to hydroxybutyrate (HB) repeating units was striking. In the cyanobacteria-dominated green material of Sippewissett mats, the mole percent ratio of repeating units was generally 1HB:1HV. In the purple sulfur bacteria-dominated pink material the relationship was typically 1HB:2HV. In Sippewissett mats, PHA contributed about 0.5 to 1\% of the organic carbon in the green layer and up to 6\% in the pink layer. In Ebro Delta mats, PHA of approximately 1HB:2HV-repeating-unit distribution contributed about 2\% of the organic carbon of the composite photosynthetic layers (the green and pink layers were not separated). Great Sippewissett Salt Marsh mats were utilized for more extensive investigation of seasonal, diel, and exogenous carbon effects. When the total PHA content was normalized to organic carbon, there was little seasonal variation in PHA levels. However, routine daily variation was evident at all sites and seasons. In every case, PHA levels increased during the night and decreased during the day. This phenomenon was conspicuous in the pink layer, where PHA levels doubled overnight. The daytime declines could be inhibited by artificial shading. Addition of exogenous acetate, lactate, and propionate induced two- to fivefold increases in the total PHA levels when applied in the daylight but had no effect when applied at night. The distinct diel pattern of in situ PHA accumulation at night appears to be related, in some phototrophs, to routine dark energy metabolism and is not influenced by the availability of organic nutrients.},
	keywords = {Alkanes, Biofilms, Hydroxybutyrates, Massachusetts, Photosynthesis, Seasons, Spain, Valerates, Water, Water Microbiology},
	issn = {0099-2240},
	author = {Rothermich, M M and Guerrero, R and Lenz, R W and Goodwin, S}
}
@article {863,
	title = {Enzymatic hydrolysis of oligomeric models of poly-3-hydroxybutyrate.},
	journal = {Biomacromolecules},
	volume = {1},
	year = {2000},
	month = {2000 Winter},
	pages = {577-83},
	abstract = {The mechanism of the enzymatic degradation of poly([R]-3-hydroxybutyrate) (PHB) was investigated by using well-defined model substrates, including both linear and cyclic [R]-3-hydroxybutyrate (3HB) and [R]-3-hydroxyvalerate (3HV) oligomers, with two different PHB depolymerases. The linear and cyclic oligomers containing from 2 to 10 repeating units were hydrolyzed in solutions of the depolymerase isolated from Aspergillus fumigatus and Alcaligenes faecalis, and the rates of hydrolysis and types of products formed were characterized. Both of the depolymerases catalyzed the hydrolysis of the cyclic oligomers (macrolides) which contained more than three 3HB and 3HV repeating units. The degradation reactions of the linear and cyclic 3HB oligomers with the A. fumigatus depolymerase gave similar ratios of monomer-to-dimer products, but PHB itself formed mostly monomer on hydrolysis, indicating that the enzymatic hydrolysis reactions occurred by different mechanisms for these different types of substrates. The results of this study conclusively show that at least the endo mode of polymer hydrolysis occurs with the two enzymes studied, while the A. fumigatus depolymerase was found to utilize both endo and exo modes of hydrolysis to efficiently degrade PHB and 3HB oligomers.},
	keywords = {3-Hydroxybutyric Acid, Alcaligenes, Aspergillus fumigatus, Carboxylic Ester Hydrolases, Escherichia coli, Hydrolysis, Kinetics, Macrolides, Models, Chemical, Polymers},
	issn = {1525-7797},
	author = {Scherer, T M and Fuller, R C and Goodwin, S and Lenz, R W}
}
@article {864,
	title = {In vitro polymerization and copolymerization of 3-hydroxypropionyl-CoA with the PHB synthase from Ralstonia eutropha.},
	journal = {Biomacromolecules},
	volume = {1},
	year = {2000},
	month = {2000 Fall},
	pages = {433-9},
	abstract = {The poly(3-hydroxybutyrate) (PHB) synthase of Ralstonia eutropha, which was produced by a recombinant strain of Escherichia coli and purified in one step with a methyl-HIC column to a purity of more than 90\%, was used to polymerize 3-hydroxypropionyl-CoA (3HPCoA) and to copolymerize 3HPCoA with 3-hydroxybutyryl-CoA (3HBCoA). A Km of 189 microM and a kcat of 10 s-1 were determined for the activity of the enzyme in the polymerization reaction of 3HPCoA based on the assumption that the dimer form of PHB synthase was the active form. Free coenzyme A was found to be a very effective competitive inhibitor for the polymerization of 3HPCoA with a Ki of 85 microM. The maximum degree of conversion of 3HPCoA to polymer was less than 40\%. In the simultaneous copolymerization reactions of these two monomers, both the turnover number for the copolymerization reaction and the maximum degree of conversion of 3HPCoA and 3HBCoA to copolymers increased with an increase in the amount of 3HBCoA in the monomer mixture. However, the maximum conversion of 3HPCoA to copolymer was always less than 35\%, regardless of the ratio of 3HPCoA to 3HBCoA. Block copolymers were obtained by the sequential copolymerization of the two monomers and these copolymers had a much narrower molecular weight distribution than those obtained by the simultaneous copolymerization for the same molar ratio of 3HPCoA to 3HBCoA.},
	keywords = {Acyltransferases, Coenzyme A, Escherichia coli, Kinetics, Magnetic Resonance Spectroscopy, Molecular Weight},
	issn = {1525-7797},
	author = {Song, J J and Zhang, S and Lenz, R W and Goodwin, S}
}
@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}
}
@article {869,
	title = {Extracellular degradation of medium chain length poly(beta-hydroxyalkanoates) by Comamonas sp.},
	journal = {Int J Biol Macromol},
	volume = {25},
	year = {1999},
	month = {1999 Jun-Jul},
	pages = {135-43},
	abstract = {The PHA-degrading isolate, strain P37C, was enriched from residential compost for its ability to hydrolyze the medium chain length PHA, poly(beta-hydroxyoctanoate) (PHO). It was subsequently found to grow on a wide range of PHAs, including both short chain length and medium chain length PHAs. The isolate was identified as belonging to the genus Comamonas. Strain P37C formed clear zones on poly(beta-hydroxybutyrate) (PHB), (PHO) and poly(beta-hydroxyphenylvalerate) (PHPV) overlay plates. PHA clear zone tubes were prepared using seven different kinds of PHAs, ranging from PHB with four-carbon repeating units, to poly(beta-hydroxyoctanoate-co-beta-hydroxyundecanoate) (PHOU) with 8- and 11-carbon repeating units. There was a direct correlation between PHA side chain length and rate of hydrolysis of the PHAs. A series of PHOUs containing varying percentages of unsaturated bonds were used to make a series of epoxidized PHOUs (PHOEs) with varying percentages of epoxy functions. Results of clear zone tube assays showed that these functionalized PHAs were all biodegradable by strain P37C, and there was no apparent correlation between rate of biodegradation and the proportion of functional groups in the PHAs. Biodegradability of these PHAs was verified using respirometry and enzyme assays. Cell-free supernatants containing activity toward PHAs were prepared, and strain P37C was shown to synthesize at least two distinct PHA depolymerases for the hydrolysis of SCL and MCL PHAs.},
	keywords = {Carboxylic Ester Hydrolases, Gram-Negative Aerobic Rods and Cocci, Kinetics, Polyesters, Structure-Activity Relationship, Substrate Specificity},
	issn = {0141-8130},
	author = {Quinteros, R and Goodwin, S and Lenz, R W and Park, W H}
}
@article {870,
	title = {Extracellular polymerization of 3-hydroxyalkanoate monomers with the polymerase of Alcaligenes eutrophus.},
	journal = {Int J Biol Macromol},
	volume = {25},
	year = {1999},
	month = {1999 Jun-Jul},
	pages = {55-60},
	abstract = {Previous investigations on the role of the polymerase in the synthesis of poly-3-hydroxybutyrate (PHB) are reviewed, and the results from earlier in vitro studies on the activity and selectivity of the polymerase of Alcaligenes eutrophus are discussed. In the present study the effect of glycerol on stabilizing the polymerase after purification and on eliminating the lag phase in in vitro polymerization reactions of 3-hydroxybutyl CoA (HBCoA), and 3-hydroxyvaleryl CoA (HVCoA) are described. K(M) values were determined for the activity of the polymerase with both HBCoA and HVCoA, and the rates of propagation for both monomers were estimated. With a racemic mixture of HBCoA, the enzyme polymerized only the [R] monomer.},
	keywords = {Acyl Coenzyme A, Acyltransferases, Alcaligenes, Hydroxybutyrates, Kinetics, Polyesters, Substrate Specificity},
	issn = {0141-8130},
	author = {Lenz, R W and Farcet, C and Dijkstra, P J and Goodwin, S and Zhang, S}
}
