Intracellular Degradation of Polyhydroxyalkanoates (PHAs)

Saito, Terumi; Kobayashi, T

doi:10.1002/3527600035.bpol3b02

Cited by 10 publications

(13 citation statements)

References 62 publications

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“…Intracellular PHB, which is accumulated in an amorphous state, is degraded by several hydrolases and the degraded products are used as a source of carbon and energy (36). When PHBaccumulating bacteria die, the PHB released into the environment is hydrolyzed by various microorganisms which secrete extracellular PHB depolymerases (12).…”

Section: Poly(3-hydroxybutyrate) (Phb) a Homopolymer Of R(ϫ)-3-mentioning

confidence: 99%

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

2005

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Section: Poly(3-hydroxybutyrate) (Phb) a Homopolymer Of R(ϫ)-3-mentioning

confidence: 99%

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

2005

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“…Only recently, three intracellular PHA depolymerases (PhaZ1, PhaZ2 and PhaZ3) and a 3HB-oligomer hydrolase (previously also designated PhaZ2), which hydrolyse PHAs and the cleavage products produced by PhaZs, were cloned and characterized in R. eutropha (Saegusa et al, 2001(Saegusa et al, , 2002Kobayashi et al, 2003;York et al, 2003). However, in contrast to the extracellular degradation of PHAs (Jendrossek et al, 1996Jendrossek & Handrick, 2002), relatively little is known about intracellular mobilization and degradation in PHA-accumulating cells (Handrick et al, 2000;Saito & Kobayashi, 2002).…”

Section: Introductionmentioning

confidence: 99%

The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha

et al. 2004

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Analysis of the genome sequence of the polyhydroxyalkanoate-(PHA) accumulating bacterium Ralstonia eutropha strain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20?0, 20?2, 19?6 and 20?2 kDa, respectively. RT-PCR analysis showed that phaP2, phaP3 and phaP4 were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts of R. eutropha had previously been shown by others. Although PhaP2 could not be localized in vivo on poly(3HB) granules, in vitro experiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues in Ralstonia solanacearum, Burkholderia fungorum and Azotobacter vinelandii. These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.

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“…1 and 3) (20). On the other hand, R. rubrum PHB depolymerase is not related to intracellular PHB depolymerases of W. eutropha and P. denitrificans in terms of amino acid homology (10,16,24,38,40,53). Therefore, R. rubrum PHB depolymerase represents a novel type of PHB depolymerase.…”

Section: Discussionmentioning

confidence: 91%

“…Many extracellular dPHA depolymerases (EC 3.1.1.75 and EC 3.1.1.76) have been characterized at the molecular level during the past decade (for a recent summary, see reference 20), but relatively little is known about nPHA depolymerases (40). Recently, an unusual extracellular nPHB depolymerase (PhaZ7) of Paucimonas lemoignei (PhaZ7), which was specific for nPHB and was unable to hydrolyze dPHB, was described (15).…”

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confidence: 99%

“…Recently, an unusual extracellular nPHB depolymerase (PhaZ7) of Paucimonas lemoignei (PhaZ7), which was specific for nPHB and was unable to hydrolyze dPHB, was described (15). For W. eutropha, the presence of several isoenzymes of intracellular nPHB depolymerases (PhaZ1 to PhaZ3) has been proposed (16,24,(38)(39)(40)53), and recently, a fourth (intracellular) PHB depolymerase gene was identified on the pHG1 megaplasmid of W. eutropha H16 (42). Intracellular nPHB depolymerases of W. eutropha are not related to extracellular dPHB depolymerases with respect to amino acid sequence but share significant amino acid similarities with each other and with other putative intracellular PHB depolymerases found in the databases (10,38,53).…”

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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

et al. 2004

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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.Polyhydroxyalkanoates (PHA) are bacterial storage compounds that can be accumulated to as much as 90% of cellular dry weight during unbalanced growth in the form of inclusion bodies (for recent reviews, see references 7 and 30). Because of their thermoplastic properties and biodegradability, PHA have attracted considerable academic and industrial interest during the last 2 decades. About 150 hydroxyalkanoic acids other than 3-hydroxybutyrate (3HB) have been identified as constituents of PHA (23,45,46). The most frequently occurring PHA in bacteria is poly(3-hydroxybutyrate) (PHB), which has been commercialized under the trade name BIOPOL. Recently, a novel class of biopolymers, with thioester linkages instead of oxoester bonds, has been found in Wautersia (Ralstonia) eutropha (50) and has been classified as polythioesters (27,28).Investigation of the biodegradation of PHA should distinguish between intracellular and extracellular degradation (for a recent review, see reference 20). Intracellular degradation is the active mobilization (hydrolysis) of the polymer by the accumulating bacterium itself. In the case of...

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Intracellular Degradation of Polyhydroxyalkanoates (PHAs)

Abstract: Introduction Historical Outline Intracellular PHA Depolymerase Endogenous Degradation of PHA Intracellular mcl‐PHA Depolymerase Intracellular Poly(3HB) Depolymerase Intracellular d (–)‐3‐Hydroxybutyrate Oligomer Hydrolase … Show more

Cited by 10 publications

References 62 publications

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia 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

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