PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

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Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative ␣/␤-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism. P olyhydroxybutyrate (PHB) and related polyhydroxyalkanoates (PHA) are storage compounds for carbon and energy and are widespread in prokaryotic species (1). PHB is deposited in the form of 200-to 500-nm particles (granules) when PHB-accumulating bacteria are cultivated in the presence of a surplus of a suitable carbon source. Ralstonia eutropha H16 (alternative designation, Cupriavidus necator) has become the model organism of PHB research, and the species is also used in industrial processes to produce PHB as a biodegradable polymer with plastic-like properties (2-4). The intensive research of the last few decades has led to a good understanding of the biochemical routes leading to PHB/PHA and of the biochemical properties of proteins with established functions in PHB/PHA metabolism. For example, the key enzymes of PHB synthesis, the PHB synthases (PhaCs), of many PHB-accumulating species have been purified; the coding genes were cloned; and the polymerization reaction has been studied (for overviews and recent results, see references 5 to 11). Meanwhile, much evidence has accumulated showing that PHB granules are not simply storage molecules but represent well-defined subcellular organelles that consist of a polymer core and a surface layer to which many proteins with specific functions are attached (12). Besides the aforementioned PHB synthase, small amphiphilic polypeptides, so-called phasin proteins (PhaP...

Section: Preparation Of Subcellular Fractions Cultures Of R Eutrophsupporting

confidence: 69%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

Sznajder

Pfeiffer

Jendrossek

2015

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“…Therefore, PhaPs and the detergents appear to have different activation mechanisms for PhaCs. Most recently, PhaM, a DNA-and PHA-binding protein, has been shown to have an activating effect for PhaC Re (26). PhaPs and PhaM have the same ability to bind PHA granules; however, their effects on PhaC Re activity were opposite.…”

Section: Discussionmentioning

confidence: 88%

Phasin Proteins Activate Aeromonas caviae Polyhydroxyalkanoate (PHA) Synthase but Not Ralstonia eutropha PHA Synthase

Ushimaru

Motoda

Numata

et al. 2014

bIn this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaC Ac ). In in vitro assays, among the three PhaCs tested, PhaC Ac was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaC Ac ), whereas the prepolymerization PhaC Re (derived from Ralstonia eutropha) and PhaC Da (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaC Ac showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C 6 ). PhaP Ac also activated PhaC Ac when it was added during polymerization (polymer-elongating PhaC Ac ), while this effect was not observed for PhaC Re . In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaP Ac expression on PHA synthesis by PhaC Ac or PhaC Re was examined. As PhaP Ac expression increased, PHA production was increased by up to 2.3-fold in the PhaC Ac -expressing strain, whereas it was slightly increased in the PhaC Re -expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaC Ac both in vitro and in vivo but do not activate PhaC Re . This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaC Ac .

“…For example, a mutant strain of Synechocystis sp. strain PCC 6803 that does not produce phasin PhaP Sp showed reduced activity of the PHB synthase (12), and phasin PhaM from R. eutropha was described as the physiological activator of the PHB synthase in this microorganism (41).…”

Section: Role Of Phasins In Pha Metabolismmentioning

confidence: 99%

Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

Mezzina

Pettinari

2016

107

Phasins are the major polyhydroxyalkanoate (PHA) granule-associated proteins. They promote bacterial growth and PHA synthesis and affect the number, size, and distribution of the granules. These proteins can be classified in 4 families with distinctive characteristics. Low-resolution structural studies and in silico predictions were performed in order to elucidate the structure of different phasins. Most of these proteins share some common structural features, such as a preponderant ␣-helix composition, the presence of disordered regions that provide flexibility to the protein, and coiled-coil interacting regions that form oligomerization domains. Due to their amphiphilic nature, these proteins play an important structural function, forming an interphase between the hydrophobic content of PHA granules and the hydrophilic cytoplasm content. Phasins have been observed to affect both PHA accumulation and utilization. Apart from their role as granule structural proteins, phasins have a remarkable variety of additional functions. Different phasins have been determined to (i) activate PHA depolymerization, (ii) increase the expression and activity of PHA synthases, (iii) participate in PHA granule segregation, and (iv) have both in vivo and in vitro chaperone activities. These properties suggest that phasins might play an active role in PHA-related stress protection and fitness enhancement. Due to their granule binding capacity and structural flexibility, several biotechnological applications have been developed using different phasins, increasing the interest in the study of these remarkable proteins.