Dataset Information

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.

ABSTRACT: 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.

SUBMITTER: Handrick R

PROVIDER: S-EPMC523223 | biostudies-literature | 2004 Nov

REPOSITORIES: biostudies-literature

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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.

Handrick René R Reinhardt Simone S Kimmig Philipp P Jendrossek Dieter D

Journal of bacteriology 20041101 21

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-len ...[more]

PMID: 15489436

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Project description: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.

Project description:Efficient hydrolysis of native poly(3-hydroxybutyrate) (nPHB) granules in vitro by soluble PHB depolymerase of Rhodospirillum rubrum requires pretreatment of nPHB with an activator compound present in R. rubrum cells (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). Edman sequencing of the purified activator (17.4 kDa; matrix-assisted laser desorption ionization-time of flight mass spectrometry) revealed identity to a hypothetical protein deduced from a partially sequenced R. rubrum genome. The complete activator gene, apdA (activator of polymer degradation), was cloned from genomic DNA, expressed as a six-His-tagged protein in recombinant Escherichia coli (M(r), 18.3 x 10(3)), and purified. The effect of ApdA on PHB metabolism was studied in vitro and in vivo. In vitro, the activity of the activator could be replaced by trypsin, but recombinant ApdA itself had no protease activity. Comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein patterns of trypsin- and ApdA-treated nPHB granules isolated from different PHB-accumulating bacteria showed that trypsin activated nPHB by removing proteins of the surface layer of nPHB regardless of the origin of nPHB, but ApdA bound to and interacted with the surface layer of nPHB in a nonproteolytic manner, thereby transforming nPHB into an activated form that was accessible to the depolymerase. In vivo, expression of ApdA in E. coli harboring the PHB biosynthetic genes, phaCBA, resulted in significant increases in the number and surface/volume ratio of accumulated PHB granules, which was comparable to the effect of phasin proteins, such as PhaP in Ralstonia eutropha. The amino acid sequence of ApdA was 55% identical to the amino acid sequence of Mms16, a magnetosome-associated protein in magnetotactic Magnetospirillum species. Mms16 was previously reported to be a GTPase with an essential function in magnetosome formation (Y. Okamura, H. Takeyama, and T. Matsunaga, J. Biol. Chem. 276:48183-48188, 2001). However, no GTPase activity of ApdA could be demonstrated. We obtained evidence that Mms16 of Magnetospirillum gryphiswaldense can functionally replace ApdA in R. rubrum. Fusions of apdA and mms16 to gfp or yfp were functionally expressed, and both fusions colocalized with PHB granules after conjugative transfer to R. rubrum. In conclusion, ApdA in vivo is a PHB-bound, phasin-like protein in R. rubrum. The function of Mms16 in magnetotactic bacteria requires further clarification.

Dataset Information

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.

Publications

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.

Similar Datasets

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