Abstract
Genes encoding 3-hydroxybutyrate oligomer hydrolase (PhaZc) and 3-hydroxybutyrate dehydrogenase (Hbd) were isolated from Paracoccus denitrificans. PhaZc and Hbd were overproduced as His-tagged proteins in Escherichia coli and purified by affinity and gel filtration chromatography. Purified His-tagged proteins had molecular masses of 31 kDa and 120 kDa (a tetramer of 29-kDa subunits). The His-tagged PhaZc hydrolyzed not only 3-hydroxybutyrate oligomers but also 3-hydroxyvalerate oligomers. The His-tagged Hbd catalyzed the dehydrogenation of 3-hydroxyvalerate as well as 3-hydroxybutyrate. When both enzymes were included in the same enzymatic reaction system with 3-hydroxyvalerate dimer, sequential reactions occurred, suggesting that PhaZc and Hbd play an important role in the intracellular degradation of poly(3-hydroxyvalerate). When the phaZc gene was disrupted in P. denitrificans by insertional inactivation, the mutant strain lost PhaZc activity. When the phaZc-disrupted P. denitrificans was complemented with phaZc, PhaZc activity was restored. These results suggest that P. denitrificans carries a single phaZc gene. Disruption of the phaZc gene in P. denitrificans affected the degradation rate of PHA.
MeSH terms
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Carboxylic Ester Hydrolases / chemistry
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Carboxylic Ester Hydrolases / genetics
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Carboxylic Ester Hydrolases / isolation & purification
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Carboxylic Ester Hydrolases / metabolism*
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Cloning, Molecular
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DNA Transposable Elements
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DNA, Bacterial / chemistry
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DNA, Bacterial / genetics
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Escherichia coli / genetics
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Gene Expression
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Genetic Complementation Test
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Hydroxybutyrate Dehydrogenase / chemistry
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Hydroxybutyrate Dehydrogenase / genetics
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Hydroxybutyrate Dehydrogenase / isolation & purification
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Hydroxybutyrate Dehydrogenase / metabolism*
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Hydroxybutyrates / metabolism*
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Molecular Sequence Data
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Molecular Weight
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Mutagenesis, Insertional
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Paracoccus denitrificans / enzymology*
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Paracoccus denitrificans / metabolism*
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Polyesters / metabolism*
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Protein Multimerization
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Protein Subunits
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Sequence Analysis, DNA
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Valerates / metabolism*
Substances
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DNA Transposable Elements
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DNA, Bacterial
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Hydroxybutyrates
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Polyesters
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Protein Subunits
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Recombinant Proteins
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Valerates
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poly-beta-hydroxybutyrate
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poly(3-hydroxyvalerate)
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Hydroxybutyrate Dehydrogenase
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Carboxylic Ester Hydrolases
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hydroxybutyrate-dimer hydrolase
Associated data
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GENBANK/AB839358
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GENBANK/AB839771
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GENBANK/AB839772