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.     

Properties of an Intracellular Poly(3-hydroxybutyrate) Depolymerase (PhaZ1) from <Emphasis Type="Italic">Rhodobacter spheroides</Emphasis>

The gene of an intracellular poly(3-hydroxybutyrate) (iPHB) depolymerase from Rhodobacter sphaeroides was cloned and sequenced. The nucleotide sequence of the cloned gene was homologous to that of the iPHB depolymerase gene from Ralstonia eutropha H16 (phaZ1 _Reu) and the gene was designated phaZ1 _Rsh. PhaZ1_Rsh was purified from E. coli harboring an expression vector containing phaZ1 _Rsh and its properties were examined. PhaZ1_Rsh degraded amorphous PHB granules, and the 3-hydroxybutyrate tetramer and pentamer, but not crystalline PHB granules. The enzyme activity was inhibited by p-chloromercuribenzoate and Triton X-100. Diisopropylfluorophosphate, phenylmethylsulfonylfluoride, and dithiothreitol had no effect on the activity. A mutant having alanine instead of cysteine at 178 lost the activity. These results show that PhaZ1_Rsh is a quite similar enzyme to PhaZ1_Reu.     

Ralstonia eutropha H16 encodes two and possibly three intracellular Poly[D-(-)-3-hydroxybutyrate] depolymerase genes

Intracellular poly[D-(-)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share approximately 30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (Delta phaZ1, Delta phaZ2, Delta phaZ3, Delta phaZ1 Delta phaZ2, Delta phaZ1 Delta phaZ3, Delta phaZ2 Delta phaZ3, and Delta phaZ1 Delta phaZ2 Delta phaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated approximately 80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.     

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.     

The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases

Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser(42), Asp(138), and His(178) were identified as catalytic-triad amino acids, with Ser(42) as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum.     

Biochemical characterization of a new type of intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic activity on native PHB granules

A Rhodospirillum rubrum gene that is predicted to code for an extracellular poly(3-hydroxybutyrate) (PHB) depolymerase by the recently published polyhydroxyalkanoates (PHA) depolymerase engineering database was cloned. The gene product (PhaZ3 _Rru ) was expressed in recombinant E. coli, purified and biochemically characterized. PhaZ3 _Rru turned out, however, to share characteristics of intracellular PHB depolymerases and revealed a combination of properties that have not yet been described for other PHB depolymerases. A fusion of PhaZ3 _Rru with the enhanced cyan fluorescent protein was able to bind to PHB granules in vivo and supported the function as an intracellular PHB depolymerase. Purified PhaZ3 _Rru was specific for short-chain-length polyhydroxyalkanoates (PHA_SCL) and hydrolysed both untreated native PHB granules as well as trypsin-activated native PHB granules to a mixture of mono- and dimeric 3-hydroxybutyrate. Crystalline (denatured) PHB granules were not hydrolysed by PhayZ3 _Rru . Low concentrations of calcium or magnesium ions (1–5 mM) reversibly (EDTA) inhibited the enzyme. Our data suggest that PhaZ3 _Rru is the representative of a new type of the growing number of intracellular PHB depolymerases.     

Properties of a novel intracellular poly(3-hydroxybutyrate) depolymerase with high specific activity (PhaZd) in Wautersia eutropha H16

A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.     

Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system.

Pseudomonas lemoignei has five different polyhydroxyalkanoate (PHA) depolymerase genes (phaZ1 to phaZ5), which encode the extracellularly localized poly(3-hydroxybutyrate) (PHB) depolymerases C, B, and D, poly(3-hydroxyvalerate) (PHV) depolymerase, and PHB depolymerase A, respectively. Four of the five genes (phaZ1 to phaZ4) have been cloned, and one of them (phaZ1) was studied in detail earlier (D. Jendrossek, B. Müller, and H. G. Schlegel, Eur. J. Biochem. 218:701-710, 1993). The fifth PHA depolymerase gene (phaZ5) was identified by colony hybridization of recombinant Escherichia coli clones with a phaZ5-specific oligonucleotide. The nucleotide sequence of a 3,704-bp EcoRI fragment was determined and found to contain two large open reading frames (ORFs) which coded for a polypeptide with significant similarities to glycerol-3-phosphate dehydrogenases of various sources (313 amino acids; M(r), 32,193) and for the precursor of PHB depolymerase A (PhaZ5; 433 amino acids; M(r), 44,906). The PHV depolymerase gene (phaZ4) was subcloned, and the nucleotide sequence of a 3,109-bp BamHI fragment was determined. Two large ORFs (ORF3 and ORF4) that represent putative coding regions were identified. The deduced amino acid sequence of ORF3 (134 amino acids; M(r), 14,686) revealed significant similarities to the branched-chain amino acid aminotransferase (IlfE) of enterobacteria. ORF4 (1,712 bp) was identified as the precursor of a PHV depolymerase (567 amino acids; M(r), 59,947). Analysis of primary structures of the five PHA depolymerases of P. lemoignei and of the PHB depolymerases of Alcaligenes faecalis and Pseudomonas pickettii revealed homologies of 25 to 83% to each other and a domain structure: at their N termini, they have typical signal peptides of exoenzymes. The adjacent catalytic domains are characterized by several conserved amino acids that constitute putative catalytic triads which consist of the consensus sequence of serine-dependent hydrolases including the pentapeptide G-X-S-X-G, a conserved histidine and aspartate, and a conserved region resembling the oxyanion hole of lipases. C terminal of the catalytic domain an approximately 40-amino-acid-long threonine-rich region (22 to 27 threonine residues) is present in PhaZ1, PhaZ2, PhaZ3, and PhaZ5. Instead of the threonine-rich region PhaZ4 and the PHB depolymerases of A. faecalis and P. pickettii contain an approximately 90-amino-acid-long sequence resembling the fibronectin type III module of eucaryotic extracellular matrix proteins. The function of the fibronectin type III module in PHA depolymerases remains obscure. Two types of C-terminal sequences apparently represent substrate-binding sites; the PHB type is present in the PHB depolymerases of A. faecalis and P. pickettii and in PhaZ2, PhaZ3, and PhaZ5 and the PHV type is present in the PHV-hydrolyzing depolymerases (PhaZ4 and PhaZ1). phaZ1 was transferred to A. eutrophus H16 and JMP222. All transconjugants of both strains were able to grow with extracellular PHB as a carbon source and produced translucent halos on PHB-containing solid media. PhaZ1, PhaZ2, PhaZ4, and PhaZ5 were purified from P. lemoignei and from recombinant E. coli; the processing sites of the precursors in E. coli were the same as in P. lemoignei, and similar substrate specificities were determined for the wild-type and the recombinant proteins. All PHA depolymerases hydrolyzed PHB at high specific activities. PhaZ1 and PhaZ4 additionally cleaved PHV, and PhaZ4 hydrolyzed poly(4-hydroxybutyrate). None of the depolymerases was able to hydrolyze polyactide or PHA consisting of monomers with more than five carbon atoms. While the wild-type depolymerase proteins were glycosylated and found to contain glucose and N-acetylglucosamine, none of the recombinant proteins was glycosylated. PHB hydrolysis was dependent on divalent cations such as Ca2+ and was inhibited by the presence of EDTA.     

Hydrolysis of native poly(hydroxybutyrate) granules (PHB), crystalline PHB, and artificial amorphous PHB granules by intracellular and extracellular depolymerases.

Native poly(hydroxybutyrate) (PHB) granules, purified PHB and artificial amorphous PHB granules were examined as putative substrates for hydrolysis by the intracellular depolymerase system of Rhodospirillum rubrum and the extracellular depolymerase of Pseudomonas lemoignei. The R. rubrum depolymerizing system requires pretreatment of granules with a heat stable 'activator' fraction; the activator can be replaced by mild trypsin treatment. Artificial granules were prepared with a cationic detergent, cetyltrimethylammonium bromide (CTAB) and an anionic detergent, (sodium cholate). Cholate and CTAB PHB granules were hydrolyzed by both enzyme systems; however, some differences were noted. Cholate granules were hydrolyzed in the absence of the R. rubrum activator fraction. Activator was required for the hydrolysis of CTAB granules but could be replaced by heparin in the extracellular depolymerase system but not in the intracellular depolymerase system. A Triton X-114 extract of native PHB granules inhibited the hydrolysis of trypsin-activated granules by the intracellular depolymerase. The inhibition was reversed by the activator fraction. Detergent extracts of granules activated with the R. rubrum activator were unable to inhibit the hydrolysis of trypsin-activated granules. These data suggest that the activator acts to modify an inhibitor present on native granules.     

Identification and Characterization of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase from Bacillus megaterium

A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of Bacillus megaterium. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the phaZ1 gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in B. megaterium, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for B. megaterium PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (R)-3HB.Polyhydroxyalkanoates (PHAs) are a group of polyesters that are produced by numerous bacteria as carbon and energy storage materials in response to nutritional stress (13, 27, 29). Poly(3-hydroxybutyrate) (PHB) is the most common and intensively studied PHA. Intracellular native PHB (nPHB) granules are composed of a hydrophobic PHB core and a surface layer consisting of proteins and phospholipids (13). The PHB of intracellular nPHB granules is in an amorphous state. When intracellular nPHB granules are exposed to extracellular environments due to cell death and lysis, the amorphous PHB is transformed into a denatured semicrystalline state. nPHB granules subjected to physical damage or solvent extraction to remove the surface layer can also crystallize into denatured PHB (dPHB) (13, 15). Artificial PHB (aPHB) granules, in which PHB is in an amorphous state, can be prepared from semicrystalline dPHB and detergents (1, 11, 23, 31).Various extracellular PHB depolymerases (PhaZs) that are secreted by many PHB-degrading bacteria have been demonstrated to specifically degrade dPHB (13, 14, 37). One exception is that PhaZ7, an extracellular PHB depolymerase secreted by Paucimonas lemoignei, displays unusual substrate specificity for amorphous PHB, with 3-hydroxybutyrate (3HB) oligomers as the main products of enzymatic hydrolysis (7). PhaZ7 exhibits no enzymatic activity toward dPHB. So far, a growing number of intracellular PHB depolymerases have been characterized. The intracellular PHB depolymerase PhaZa1 of Ralstonia eutropha (also called Cupriavidus necator) H16 has recently been established to be especially important for the intracellular mobilization of accumulated PHB (42). The main in vitro hydrolytic products of PhaZa1 degradation of amorphous aPHB are 3HB oligomers (31). PhaZd1, another intracellular PHB depolymerase of R. eutropha H16, shows no significant amino acid similarity to PhaZa1. The in vitro hydrolytic products of PhaZd1 degradation of amorphous aPHB are also 3HB oligomers. A 3HB monomer is rarely detected as a hydrolytic product (1). The intracellular PHB depolymerase PhaZ of Paracoccus denitrificans was reported previously to degrade protease-treated nPHB granules in vitro, with the release of 3HB dimers and oligomers as the main hydrolytic products (6). Recently, we have identified a novel intracellular PHB depolymerase from Bacillus thuringiensis serovar “israelensis” (39). The B. thuringiensis PhaZ shows no significant amino acid similarity to any known PHB depolymerase. This PhaZ has strong amorphous PHB-hydrolyzing activity and can release a considerable amount of 3HB monomers by the hydrolysis of trypsin-treated nPHB granules (39). It is of note that purified PhaZd1 from R. eutropha, PhaZ from P. denitrificans, and PhaZ from B. thuringiensis need pretreatment of nPHB granules with protease to remove surface proteins for PHB degradation (1, 6, 39). They show only very little or no activity toward nPHB granules without trypsin pretreatment. It has been demonstrated previously that these intracellular PHB depolymerases cannot hydrolyze dPHB (1, 31, 39).(R)-3HB, a biotechnologically valuable chiral compound, has been widely used for syntheses of antibiotics, vitamins, and pheromones (3, 30, 38). One way to produce (R)-3HB is heterologous coexpression of a PHB synthetic operon and a gene encoding an amorphous PHB-degrading PhaZ in Escherichia coli (3, 18, 25, 33, 38). A common problem encountered by this method is that oligomeric and dimeric forms of 3HB often constitute a major portion of the products of enzymatic hydrolysis, thus requiring further hydrolysis by 3HB oligomer hydrolase or heating under alkaline conditions to generate 3HB monomers (3, 18, 25, 33).Bacillus megaterium genes involved in the biosynthesis of nPHB granules have been cloned from strain ATCC 11561 and characterized previously (19, 21, 22). A gene encoding the extracellular PHB depolymerase PhaZ from B. megaterium was recently cloned from strain N-18-25-9 (34). However, little is known about B. megaterium genes involved in the intracellular mobilization of PHB. In this study, we have identified in B. megaterium ATCC 11561 an intracellular PHB depolymerase that could rapidly degrade nPHB granules in vitro without the need for trypsin pretreatment of the nPHB granules. Moreover, almost all the in vitro hydrolytic products released from the degradation of amorphous PHB by this PhaZ were 3HB monomers. This PhaZ could also hydrolyze dPHB with the generation of 3HB monomers. Thus, it appears to be a novel intracellular PHB depolymerase and may have promising potential for biotechnological application in the production of enantiomerically pure (R)-3HB monomers.     

Poly(3-hydroxyvalerate) depolymerase of Pseudomonas lemoignei

Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA(SCL)) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596-607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA(SCL) depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [M(r)], 43,610 Da) resembles precursors of other extracellular PHA(SCL) depolymerases (28 to 50% identical amino acids). The mature protein (M(r), 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S(136), D(211), and H(269) similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA(SCL) depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA(SCL) depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA(SCL) depolymerases.     

Isolation of a Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterium from compost, and cloning and characterization of a gene encoding PHB depolymerase of Bacillus megaterium N-18-25-9

A Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterial strain was isolated from compost. This organism, identified as Bacillus megaterium N-18-25-9, produced a clearing zone on opaque NB-PHB agar, indicating the presence of extracellular PHB depolymerase. A PHB depolymerase gene, PhaZ(Bm), of B. megaterium N-18-25-9 was cloned and sequenced, and the recombinant gene product was purified from Escherichia coli. The N-terminal half region of PhaZ(Bm) shared significant homologies with a catalytic domain of other PHB depolymerases. Although the C-terminal half region of PhaZ(Bm) showed no significant similarity with those of other PHB depolymerases, that region was necessary for the PHB depolymerase activity. Therefore, this enzyme's domain structure is unique among extracellular PHB depolymerase domain structures. The addition of PHB to the medium led to a sixfold increase in PhaZ(Bm) mRNA, while the presence of glucose repressed PhaZ(Bm) expression. The maximum activity was observed at pH 9.0 at 65 degrees C.     

The "PHB depolymerase inhibitor" of Paucimonas lemoignei is a PHB depolymerase

A approximately 35 kDa protein that has been described to be secreted by Paucimonas lemoignei during growth on succinate and to inhibit hydrolysis of denatured (crystalline) poly(3-hydroxybutyrate) (dPHB) by extracellular PHB depolymerases of P. lemoignei (PHB depolymerase inhibitor (PDI)) was purified and characterized. Purified PDI (M(r), 36 199 +/- 45 Da) inhibited hydrolysis of dPHB by two selected purified PHB depolymerases (PhaZ2 and PhaZ5) but did not inhibit the hydrolysis of water-soluble substrates such as p-nitrophenylbutyrate by PhaZ5 and PhaZ2. PDI revealed a high binding affinity to dPHB although it was not able to hydrolyze the crystalline polymer. However, purified PDI had a high hydrolytic activity if native (amorphous) PHB (nPHB) was used as a substrate. N-terminal sequencing of PDI revealed that it was identical to recently described extracellular PHB depolymerase PhaZ7 which is specific for nPHB and which cannot hydrolyze dPHB. To confirm that the inhibition of hydrolysis of dPHB by PhaZ7 is an indirect surface competition effect at high depolymerase concentration, the activity of PHB depolymerases PhaZ2 and PhaZ5 in the presence of different amounts of protein mixtures was determined. The components of NB or LB medium inhibited hydrolysis of the polymer in a concentration-dependent manner but had no effect on the hydrolysis of p-nitrophenylbutyrate by PHB depolymerases. In combination with PHB depolymerases PhaZ2 and PhaZ5 the protein PhaZ7 ("PDI") enables the bacteria to hydrolyze dPHB and nPHB simultaneously.     

Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei

The recently discovered extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser136-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids. In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800. The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser136Cys) were expressed and efficiently secreted by B. subtilis as shown by Western blots of cell-free culture fluid proteins. No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His47Ala, Ser136Ala, Asp242Ala, Asp242Asn, His306Ala, indicating the importance of these amino acids for activity. Replacement of Ser136 by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine. Alterations of Asp256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser136-Met-Gly) to a lipase box consensus sequence (Gly134-His-Ser136-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly134-Leu135-Ser136-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity. In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His47, Ser136, Asp242 and His306.     

Poly(3-Hydroxyvalerate) Depolymerase of Pseudomonas lemoignei

Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA_SCL) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596–607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA_SCL depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [M_r], 43,610 Da) resembles precursors of other extracellular PHA_SCL depolymerases (28 to 50% identical amino acids). The mature protein (M_r, 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S₁₃₆, D₂₁₁, and H₂₆₉ similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA_SCL depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA_SCL depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA_SCL depolymerases.     

Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha

Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilized R. eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R. eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M

Abstract Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zooglea ramigera I-16-M were examined. The release of d (−)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of d (−)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing d (−)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

Poly(3-hydroxybutyrate) (PHB) depolymerase from Alcaligenes faecalis T1 is composed of three domains: the catalytic (C) domain, the fibronectin type III-like (F) domain, and the substrate-binding (S) domain. We constructed domain deletion, inversion, chimera, and extra-F-domain mutants and examined their enzyme activity and PHB-binding ability. In addition, we performed substitution of 214Asp and 273His with glycine and aspartate, respectively, to examine their participation in a catalytic triad together with 139Ser. The mutant with both the F and S domains deleted and the trypsin-digested enzyme showed no PHB-hydrolyzing activity and less PHB-binding ability than that of the wild-type enzyme but retained D-(-)-3-hydroxybutyrate trimer-hydrolyzing activity at a level similar to that of the wild-type enzyme. The mutant with the F domain deleted and the mutant which had the order of the F and S domains inverted retained PHB-binding ability and trimer-hydrolyzing activity at levels similar to those of the wild-type enzyme but lost PHB-hydrolyzing activity. The chimera mutant, in which the F domain was substituted with a Thr-rich domain of PHB depolymerase A from Pseudomonas lemoignei, and the extra-F-domain mutant, with an additional F domain, retained trimer- and PHB-hydrolyzing activities and PHB-binding ability at levels similar to those of the wild-type enzyme. Two mutants (D214G and H273D) showed no enzymatic activity toward trimer and PHB, and they were not labeled with [3H]diisopropylfluorophosphate.     

Impact of Ralstonia eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB Storage in Recombinant Escherichia coli

The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay of these enzymes.     

A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyesters of short chain-length hydroxyalkanoic acids.

A novel type of hydrolase was purified from culture fluid of Paucimonas (formerly Pseudomonas) lemoignei. Biochemical characterization revealed an unusual substrate specificity of the purified enzyme for amorphous poly((R)-3-hydroxyalkanoates) (PHA) such as native granules of natural poly((R)-3-hydroxybutyrate) (PHB) or poly((R)-3-hydroxyvalerate) (PHV), artificial cholate-coated granules of natural PHB or PHV, atactic poly((R,S)-3-hydroxybutyrate), and oligomers of (R)-3-hydroxybutyrate (3HB) with six or more 3HB units. The enzyme has the unique property to recognize the physical state of the polymeric substrate by discrimination between amorphous PHA (good substrate) and denatured, partially crystalline PHA (no substrate). The pentamers of 3HB or 3HV were identified as the main products of enzymatic hydrolysis of native PHB or PHV, respectively. No activity was found with any denatured PHA, oligomers of (R)-3HB with five or less 3HB units, poly(6-hydroxyhexanoate), substrates of lipases such as tributyrin or triolein, substrates for amidases/nitrilases, DNA, RNA, casein, N-alpha-benzoyl-l-arginine-4-nitranilide, or starch. The purified enzyme (M(r) 36,209) was remarkably stable and active at high temperature (60 degrees C), high pH (up to 12.0), low ionic strength (distilled water), and in solvents (e.g. n-propyl alcohol). The depolymerase contained no essential SH groups or essential disulfide bridges and was insensitive to high concentrations of ionic (SDS) and nonionic (Triton and Tween) detergents. Characterization of the cloned structural gene (phaZ7) and the DNA-deduced amino acid sequence revealed no homologies to any PHB depolymerase or any other sequence of data banks except for a short sequence related to the active site serine of serine hydrolases. A classification of the enzyme into a new family (family 9) of carboxyesterases (Arpigny, J. L., and Jaeger, K.-E. (1999) Biochem. J. 343, 177-183) is suggested.