title: Characterization of binding preference of polyhydroxyalkanoate biosynthesis-related multifunctional protein PhaM from Ralstonia eutropha.
abstract: The binding preference of a polyhydroxyalkanoate (PHA) biosynthesis-related multifunctional protein from Ralstonia eutropha (PhaMRe) was characterized. In vitro activity assay showed that PHA synthase from R. eutropha (PhaCRe) was activated by the presence of PhaMRe but PHA synthase from Aeromonas caviae (PhaCAc) was not. Additionally, in vitro assays of protein-protein interactions demonstrated that PhaMRe interacted with PhaCRe directly, but did not interact with PhaCAc. These results suggest that the protein-protein interaction is important for the activation of PhaC by PhaMRe. Further analyses indicated that PhaMRe has little or no direct interaction with the PHA polymer chain. Subsequently, PHA biosynthesis genes (phaA Re, phaB Re, and phaC Re/phaC Ac) and the phaM Re gene were introduced into recombinant Escherichia coli and cultivated for PHA accumulation. Contrary to our expectations, the expression of PhaMRe decreased PHA accumulation and changed the morphology of PHA granules to be microscopically obscure shape in PhaCRe-expressing E. coli. No change in the amount of P(3HB) or the morphology of granules by PhaMRe expression was observed in PhaCAc-expressing E. coli. These observations suggest that PhaMRe affects cellular physiology through the PhaM-PhaC interaction.
authors.affiliation: Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8502, Japan. pt-ushimaru@fpu.ac.jp.|Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui, 910-1195, Japan. pt-ushimaru@fpu.ac.jp.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Ushimaru
authors.affiliation: Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8502, Japan.
authors.forename: Takeharu
authors.initials: T
authors.lastname: Tsuge
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D057585:Aeromonas caviae / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D020118:Cupriavidus necator / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis*; D011485:Protein Binding; D014176:Protein Biosynthesis; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D054813:Polyhydroxyalkanoates; D011994:Recombinant Proteins; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase
keywords: PHA granule; PHA synthase; PhaM; Polyhydroxyalkanoate; Protein-protein interaction