Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

Gebauer, Birgit; Jendrossek, Dieter

doi:10.1128/aem.01184-06

Cited by 61 publications

(57 citation statements)

References 62 publications

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“…The bacterial strains and plasmids used in this study are listed in Table 1. R. eutropha was grown in nutrient broth (NB) or mineral salts medium with sodium gluconate (or fructose) as described previously at 30°C (1,5). Escherichia coli strains were maintained in Luria-Bertani broth (37°C).…”

Section: Methodsmentioning

confidence: 99%

“…nPHB granules were isolated from French presslysed cells (in the presence of 1 mM dithiothreitol [DTT]) by two glycerol gradient centrifugations as described previously (5,12,23). Cholate-coated artificial PHB (aPHB) granules were prepared as described elsewhere (1).…”

Section: Methodsmentioning

confidence: 99%

“…Isolated nPHB granules were examined for PHB depolymerase activity by pH stat analysis as described previously (5). Wild-type granules isolated from gluconate-grown cells had a specific acid release rate of 4 nmol min Ϫ1 mg nPHB Ϫ1 .…”

Section: Phaza1 Is a Phb Granule-bound Protein In Vivomentioning

confidence: 99%

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Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA

et al. 2007

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, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (proteinfree) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD ؉ , indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD ؉ / NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.Poly(3-hydroxybutyrate) (PHB) is the most prominent member of the bacterial polyhydroxyalkanoates (PHA). PHA are osmotic neutral reservoirs of carbon and energy and are synthesized when more carbon sources are available than can be consumed by the bacteria. PHA are reutilized (mobilized) during times of starvation and greatly enhance the survival of bacteria in the absence of a suitable exogenous carbon source.PHB is synthesized by condensation of two molecules of acetyl coenzyme A (CoA) to acetoacetyl-CoA (with a thiolase encoded by phaA), subsequent reduction to 3-hydroxybutyrylCoA (with a reductase encoded by phaB), and polymerization to PHB (with a synthase encoded by phaC). The polymer can be hydrolyzed to 3-hydroxybutyrate by PHB depolymerases (PhaZs). Biosynthesis and biodegradation of PHA have been investigated by many research groups for about three decades, and a series of books and reviews have been published (11,13,30,34,(39)(40)(41)(42). PHB exists in two different forms. In vivo PHB granules consist of an amorphous polymer and are covered by a dense layer consisting of mainly proteins (phasins, PHB synthase, PHB depolymerases, and other proteins) (14,22,31,38). Such granules are called native PHB (nPHB) granules and can be isolated in the native form by glycerol density gradient centrifugation (12,23,45). Isolated PHB granules that have been treated with solvents or with compounds that remove the surface layer rapidly crystallize and are referred to as denatured PHB granules (23,24). (For more details on the impact of the biophysical state of the polymer granules on their susceptibility to enzymatic hydrolysis see references 5, 8, and 12.) Despite the progress made in understanding the function of individual proteins involved in PHA metabolism, the molecular tools and mechanisms with which a cell decides whether it should synthesize or degrade (mobilize) PHB are not known. Several reports have indicated that PHB synthesis and PHB degradation can happen simultaneously in Ralstonia eutropha, the model organism for PHB...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA

et al. 2007

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“…Interestingly, the gene coding for the iPHA MCL depolymerase (phaZ) is located between two copies of PHA MCL synthase genes (phaC1 and phaC2) in all investigated PHA MCLaccumulating bacteria. Recently, iPHA depolymerase activity could be demonstrated in vitro by use of radiolabeled substrates (nPHA MCL ) or by pH stat (nPHA SCL ), respectively (5,16). iPHB depolymerases of PHA SCL -accumulating bacteria were not described before 2000, but in the first 5 years of this century no fewer than seven putative iPHB depolymerases and two 3HB oligomer hydrolases have been postulated for R. eutropha (1, 23, 44-46, 71, 88, 123).…”

Section: Ipha Depolymerasesmentioning

confidence: 99%

Polyhydroxyalkanoate Granules Are Complex Subcellular Organelles (Carbonosomes)

2009

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Polyhydroxyalkanoates (PHAs) such as poly(3-hydroxybutyrate) (PHB) or poly(3-hydroxyoctanoate), are universal prokaryotic storage compounds of carbon and energy. PHAs are accumulated intracellularly in form of inclusion bodies (PHA granules) during times of oversupply with carbon sources (for reviews, see references 2, 54, 64, 76, 86, and 100). PHAs can consist of short-chain-length hydroxyalkanoic acids (PHA SCL ) or medium-chain-length monomers (PHA MCL ), depending on strain and culture conditions. Given a number of more than 150 identified hydroxyalkanoates as potential constituents (101) the theoretical number of different PHA copolymers is incredibly high. A few PHAs, such as PHB and copolymers of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate, and/or 4-hydroxybutyrate, are produced by industry (Biocycle, Biomer, Biopol, Enmat, Mirel, and Nodax).The number of publications on PHA metabolism has considerably increased in the last two decades, and many aspects of the biosynthesis, molecular architecture, intracellular mobilization, extracellular degradation, and commercial applications of PHAs have been addressed. One exiting outcome of these studies was the finding that many polypeptides are specifically present on the surface of PHA granules, much more than would be essential for PHA synthesis. These proteins constitute a particular surface layer on PHA granules that is essential for PHA metabolism. In conclusion, PHA granules are complexly organized subcellular structures and appear to be more than simple polymer inclusions. The current knowledge of the biochemical functions of PHA-associated proteins will be reviewed here. This will be done using the example of Ralstonia eutropha H16, which is the model organism of PHA research and has become an important prokaryotic strain for several biotechnological applications (82). It should be noted that several other taxonomic names of R. eutropha are found in literature. These include Hydrogenomonas eutropha, Alcaligenes eutrophus, Wautersia eutropha, and Cupriavidus necator. We used here the most commonly accepted name, R. eutropha, which has been also used for description of the recently sequenced genome (71). When appropriate, the properties of PHA-bound proteins of other organisms are discussed afterward. PHA SYNTHASESPHA synthase is the key enzyme of PHA synthesis and catalyzes the polymerization of hydroxyacyl-coenzyme A (CoA) to PHA and free CoA. The first PHA synthase gene (phaC) was cloned 20 years ago. Remarkably, this was done independently in three labs with the same bacterial strain, R. eutrophus H16 (66, 95, 97). Meanwhile, several PHA synthases from different sources have been biochemically investigated, and many more PHA synthase genes have been cloned or have been determined by genome sequencing. PHA synthases currently are divided into four classes depending on their subunit composition and substrate specificity. The R. eutropha and Pseudomonas putida (previously Pseudomonas oleovorans) PHA synthases represent class I (producing PHA SCL ) and class ...

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“…The first degradation assays carried out with such isolated nPHB granules revealed the release of 3-hydroxybutyrate (3HB) in a hydrolytic reaction, although only small amounts could be detected (47). In contradiction to that study, the same laboratory revealed in 2007 a thiolytic activity of the PHB depolymerase PhaZ1.…”

mentioning

confidence: 87%

Poly(3-Hydroxybutyrate) Degradation in Ralstonia eutropha H16 Is Mediated Stereoselectively to (S)-3-Hydroxybutyryl Coenzyme A (CoA) via Crotonyl-CoA

Eggers

Steinbüchel

2013

Journal of Bacteriology

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bDegradation of poly(3-hydroxybutyrate) (PHB) by the thiolytic activity of the PHB depolymerase PhaZ1 from Ralstonia eutropha H16 was analyzed in the presence of different phasins. An Escherichia coli strain was constructed that harbored the genes for PHB synthesis (phaCAB), the phasin PhaP1, and the PHB depolymerase PhaZ1. PHB was isolated in the native form (nPHB) from this recombinant E. coli strain, and the in vitro degradation of the polyester was examined. Degradation resulted in the formation of the expected 3-hydroxybutyryl coenzyme A (3HB-CoA) and in the formation of a second product, which occurred in significantly higher concentrations than 3HB-CoA. This second product was identified by liquid chromatography mass spectrometry (LC-MS) as crotonyl-CoA. Replacement of PhaP1 by PhaP2 or PhaP4 resulted in a lower degradation rate, whereas the absence of the phasins prevented the degradation of nPHB by the PHB depolymerase PhaZ1 almost completely. In addition, the in vitro degradation of nPHB granules isolated from R. eutropha H16 (wild type) and from the R. eutropha ⌬phaP1 and ⌬phaP1-4 deletion mutants was examined. In contrast to the results obtained with nPHB granules isolated from E. coli, degradation of nPHB granules isolated from the wild type of R. eutropha yielded high concentrations of 3HB-CoA and low concentrations of crotonyl-CoA. The degradation of nPHB granules isolated from the ⌬phaP1 and ⌬phaP1-4 deletion mutants of R. eutropha was significantly reduced in comparison to that of nPHB granules isolated from wild-type R. eutropha. Stereochemical analyses of 3HB-CoA revealed that the (R) stereoisomer was collected after degradation of granules isolated from E. coli, whereas the (S) stereoisomer was collected after degradation of granules isolated from R. eutropha. Based on these results, a newly observed mechanism in the degradation pathway for PHB in R. eutropha is proposed which is connected by crotonyl-CoA to the ␤-oxidation cycle. According to this model, the NADPH-dependent synthesis of PHB with (R)-3HB-CoA as the intermediate and the PHB degradation yielding (S)-3HB-CoA, which is further converted in an NAD-dependent reaction, are separated. P oly(3-hydroxybutyrate) (PHB) is a short-carbon-chainlength polyester belonging to the bacterial polyhydroxyalkanoates (PHAs), which are formed by a wide range of Bacteria and Archaea and comprise a large variety of constituents (1-3). PHAs are stored as granules in the cytoplasm when one nutrient is limited and a carbon source is provided in excess. They serve as carbon and energy storage compounds and are degraded if no carbon source is available and if the limiting element is provided again (4). Due to their thermoplastic properties and biodegradability, PHAs are of great interest for industry (5-7).The model organism for PHB metabolism is the Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha H16, which was isolated in 1961 (8-10). About 50 years of research unraveled many aspects of the synthesis and composition of PHB. T...

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Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

Cited by 61 publications

References 62 publications

Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA

Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA

Polyhydroxyalkanoate Granules Are Complex Subcellular Organelles (Carbonosomes)

Poly(3-Hydroxybutyrate) Degradation in Ralstonia eutropha H16 Is Mediated Stereoselectively to (S)-3-Hydroxybutyryl Coenzyme A (CoA) via Crotonyl-CoA

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