Mari Shiraki

Cloning of an Intracellular Poly[d(−)-3-Hydroxybutyrate] Depolymerase Gene from Ralstonia eutropha H16 and Characterization of the Gene Product

An intracellular poly[D(-)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned from Ralstonia eutropha H16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric D(-)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutropha whose phaZ gene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha.

Purification and Properties of an Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase (PhaZ2) in Ralstonia eutropha H16 and Its Identification as a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase

An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2(Reu)) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2(Reu). The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1(Reu)). The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca. 5% in the stationary phase. A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R. eutropha along with PhaZ1.

Cloning of an Intracellular D(-)-3-Hydroxybutyrate-Oligomer Hydrolase Gene from Ralstonia eutropha H16 and Identification of the Active Site Serine Residue by Site-Directed Mutagenesis.

An intracellular D(-)-3-hydroxybutyrate (3HB)-oligomer hydrolase gene from Ralstonia eutropha (formerly Alcaligenes eutrophus) H16 was cloned, sequenced, and characterized. As a hybridization probe to screen restriction digests of chromosomal DNA, an extracellular 3HB-oligomer hydrolase gene from Ralstonia pickettii strain (formerly Pseudomonas sp. strain) A1 was used. A specific hybridization signal was obtained and a 6.5-kbp SmaI fragment was cloned in an Escherichia coli phagemid vector. The crude extract from E. coli with this plasmid showed 3HB-trimer hydrolase activity. The subcloned 3.2-kbp fragment still showed 3HB-trimer hydrolase activity in E. coli and expressed an approximately 78-kDa protein in an in vitro transcription-translation system. Nucleotide sequence analysis of the 3.2-kbp fragment showed an open reading frame that encodes a polypeptide with a deduced molecular weight of 78,510. The putative amino acid sequence showed 54% identity with that of the oligomer hydrolase from R. pickettii A1. By site-directed mutagenesis, a novel amino acid sequence (S-V-S*-N-G) containing an essential serine residue in the catalytic center of the enzyme was determined. The gene product was found in PHB-rich cells of R. eutropha by immunodetection. The expressed 3HB-oligomer hydrolase localized both in the supernatant fraction and the PHB granules of the cells.

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases

Five extracellular PHB depolymerases of bacteria isolated from various sources were purified to electrophoretic homogeneity and compared with known extracellular PHB depolymerase fromAlcaligenes faecalis T1. The molecular mass of these enzymes were all around 40–50 kDa. Nonionic detergent, diisopropylfluorophosphate and dithiothreitol inhibited the PHB depolymerase activity of all these enzymes. Trypsin abolished PHB depolymerase activity, but not theD-3-hydroxybutyric acid dimer hydrolase activity of all the enzymes. These results showed that the basic properties of these PHB depolymerases resemble those of theA. faecalis T1 enzyme. Analysis ofN-terminal amino acid sequence of the purified enzymes revealed that these enzymes includingA. faecalis T1 enzyme fall into three groups.

Properties of a Poly(3-hydroxybutyrate) Depolymerase from Penicillium funiculosum

A poly(3-hydroxybutyrate) (PHB) depolymerase was purified from a fungus, Penicillium funiculosum (IFO6345), with phenyl-Toyopearl and its properties were compared with those of other PHB depolymerases. The molecular mass of the purified enzyme was estimated at about 33 kDa by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The pH optimum and pI were 6.5 and 6.5, respectively. The purified protein showed affinity to Con A-Sepharose, indicating that it is a glycoprotein. Diisopropylfluorophosphate and dithiothreitol inhibited the depolymerase activity completely. The N-terminal amino acid sequence of the purified enzyme was TALPAFNVNPNSVSVSGLSSGGYMAAQL, which contained a “lipase box” sequence. This purified enzyme is one of the extracellular PHB depolymerase which belong to serine esterase. The purified enzyme showed relatively strong hydrolytic activity against 3-hydroxybutyrate oligomers compared with its PHB-degrading activity. PHB-binding experiments showed that P. funiculosum depolymerase has the weakest affinity for PHB of all the depolymerases examined.

Roles of poly(3-hydroxybutyrate) depolymerase and 3HB-oligomer hydrolase in bacterial PHB metabolism

Many poly-3-hydroxybutyrate (PHB)-degrading enzymes have been studied. But biological roles of 3HB-oligomer hydrolases (3HBOHs) and how PHB depolymerases (PHBDPs) and 3HBOHs cooperate in PHB metabolism are not fully elucidated. In this study, several PHBDPs and 3HBOHs from three types of bacteria were purified, and their substrate specificity, kinetic properties, and degradation products were investigated. From the results, PHBDP and 3HBOH seemed to play a role in PHB metabolism in three types of bacteria, as follows: (A) In Ralstonia pickettii T1, an extracellular PHBDP degrades extracellular PHB to various-sized 3HB-oligomers, which an extracellular 3HBOH hydrolyzes to 3HB-monomers. (B) In Acidovorax sp. SA1, an extracellular PHBDP hydrolyzes extracellular PHB to small 3HB-oligomers (dimer and trimer), which an intracellular 3HBOH efficiently degrades to 3HB in the cell

Biochemical and Genetic Characterization of a D(-)-3-Hydroxybutyrate Dehydrogenase from Acidovorax sp. Strain SA1

D(-)-3-hydroxybutyrate dehydrogenase (BDH; EC 1.1.1.30) from a poly(D(-)-3-hydroxybutyrate) (PHB) degrading bacterium, Acidovorax sp. SA1, was purified using Toyopearl DEAE-650M, red-Sepharose CL-4B, and Q Sepharose FF. The molecular mass of the enzyme was estimated as 27 kDa by SDS-PAGE and 110 kDa by gel filtration. The gene encoding BDH was cloned and sequenced, and expressed in Escherichia coli. The gene product was purified in two steps with a high yield. The N-terminal amino acid sequence of the enzyme purified from E. coli agreed with that of the purified enzyme from strain SA1. The BDH of strain SA1 had high amino acid sequence homology to that of Ralstonia eutropha H16. The Km values for D(-)-3-hydroxybutyrate and NAD+ in the oxidation reaction were 4.5 x 10(-4) M and 8.9 x 10(-5) M, respectively. The Km values for acetoacetate and NADH in the reduction reaction were 2.4 x 10(-4) M and 2.9 x 10(-5) M, respectively.

Secretion pathway for the poly(3-hydroxybutyrate) depolymerase in Ralstonia pickettii T1

The extracellular poly(3-hydroxybutyrate) depolymerase from Ralstonia pickettii T1 has been purified, its function and character investigated in detail, and its gene cloned and sequenced. However, the mechanism by which this enzyme is secreted has not been elucidated. A mutant unable to degrade poly(3-hydroxybutyrate), N17, was obtained with the random insertion of a mini-transposon, Tn5. Western analysis using antiserum against the poly(3-hydroxybutyrate) depolymerase of Ralstonia pickettii T1, revealed that N17 accumulated the poly(3-hydroxybutyrate) depolymerase in the periplasm and cytoplasm, and did not secrete the enzyme into the external medium. It was also found that 3-hydroxybutyrate-oligomer hydrolase was secreted but inactive. The disrupted gene in N17, depO, was analyzed by Southern hybridization and its nucleotide sequence was determined. One complete open reading frame was found in the cloned 2.3-kbp DNA fragment. From a BLAST search, this gene product was found to be homologous to PulO of Ralstonia eutropha JMP134 (60% identity) and XcpA of Pseudomonas aeruginosa (60% identity). These proteins are prepilin peptidase/N-metyltransferases, a component of the Type II secretion pathway. DepO also had the four cysteines highly conserved in most prepilin peptidases at the same positions. The transcript of depO was examined by Northern hybridization using depO as a probe. In the total RNA of Ralstonia pickettii T1 in the early stationary phase, a band at 2.6-kb was detected, suggesting depO to be a functional gene. In this study, it was found that poly(3-hydroxybutyrate) depolymerase was secreted by the Type II pathway.

Cloning and Sequencing of an Intracellular D(-)-3-Hydroxybutyrate Oligomer Hydrolase from Acidovorax sp. Strain SA1 and Purification of the Enzyme

The gene of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase (i3HBOH) was cloned and sequenced from a poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Acidovorax sp. strain SA1. The i3HBOH gene has 876 nucleotides corresponding to the deduced sequence of 292 amino acids. In this amino acid sequence, the general lipase box sequence (G-X1-S-X2-G) was found, whose serine residue was determined to the active sites serine by site-directed mutagenesis.An i3HBOH was purified to electrophoretical homogeneity from SA1. The molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE. The N-terminal amino acid sequence of the purified enzyme corresponded to the deduced N-terminal amino acid sequence in the cloned i3HBOH gene.This is the first cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase gene to date.

Characterization of a novel 3-hydroxybutyrate dehydrogenase from Ralstonia pickettii T1

We previously reported that the activities of two 3-hydroxybutyrate dehydrogenases (BDH1 and BDH2) were greatly influenced by culture conditions when Ralstonia pickettii T1, a strain growing on extracellular poly-3-hydroxybutyrate (PHB), was grown on different carbon sources such as 3HB and succinate. In this study, knockout mutants of bdh1 or bdh2 were constructed and characterized under different culture conditions. In addition, a novel BDH (BDH3) was found in bdh2 mutants, and bdh3 was cloned. Apparent kinetic parameters for the substrates of BDH3 indicated that the enzyme is suitable for the oxidation reaction of 3-hydroxybutyrate (3HB) to acetoacetate. In Western blotting, it was clear that BDH3 is produced only in cells grown on 3HB or PHB as a carbon source, while BDH1 and BDH2 are produced in cells grown on various carbon sources such as sugars, amino acids, organic acids, 3HB, and PHB. Both the bdh1 and bdh2 mutants lagged behind the wild type in growth rates when the cells were cultured with 3HB, citrate, succinate, or nutrient broth. A test of sensitivity to diamide as an oxidative stress revealed that the lack of BDH1 or BDH2 caused a decline in the capacity to neutralize the stress. These results suggested that BDH1 and BDH2 are needed to regulate the cytoplasmic redox state as well as to utilize 3HB, while BDH3 is specialized to utilize 3HB. The expression of bdh3 may be coordinately regulated with a gene encoding putative 3HB permease.