title: 3-Hydroxybutyrate oligomer hydrolase and 3-hydroxybutyrate dehydrogenase participate in intracellular polyhydroxybutyrate and polyhydroxyvalerate degradation in Paracoccus denitrificans.
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.
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Japan.
authors.forename: Jing
authors.initials: J
authors.lastname: Lu
authors.forename: Akira
authors.initials: A
authors.lastname: Takahashi
authors.forename: Shunsaku
authors.initials: S
authors.lastname: Ueda
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005816:Genetic Complementation Test; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000378:metabolism*; D011091:Polyesters / Q000378:metabolism*; D055503:Protein Multimerization; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014631:Valerates / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
chemical_list: D002265:Carboxylic Ester Hydrolases; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D006884:Hydroxybutyrate Dehydrogenase; D006885:Hydroxybutyrates; D011091:Polyesters; D021122:Protein Subunits; D011994:Recombinant Proteins; D014631:Valerates; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C073314:poly(3-hydroxyvalerate); C032152:hydroxybutyrate-dimer hydrolase