title: PhaM is the physiological activator of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) in Ralstonia eutropha.
abstract: Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-D-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.
authors.affiliation: Institute of Microbiology, University of Stuttgart, Stuttgart, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Pfeiffer
authors.forename: Dieter
authors.initials: D
authors.lastname: Jendrossek
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002219:Carbamates / Q000737:chemistry; D020118:Cupriavidus necator / Q000378:metabolism*; D003902:Detergents / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry; D005976:Glutaral / Q000737:chemistry; D006885:Hydroxybutyrates / Q000378:metabolism; D008970:Molecular Weight; D011091:Polyesters / Q000378:metabolism; D055503:Protein Multimerization; D000094122:Polyhydroxybutyrates
chemical_list: D000214:Acyl Coenzyme A; D000217:Acyltransferases; D001426:Bacterial Proteins; D002219:Carbamates; D003902:Detergents; D005960:Glucosides; D005976:Glutaral; D006885:Hydroxybutyrates; D011091:Polyesters; C060539:6-O-(N-heptylcarbamoyl)methylglucoside; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C030372:3-hydroxybutyryl-coenzyme A; C060970:poly-beta-hydroxybutyrate polymerase