title: Insights into the surface topology of polyhydroxyalkanoate synthase: self-assembly of functionalized inclusions.
abstract: The polyhydroxyalkanoate (PHA) synthase catalyzes the synthesis of PHA and remains attached to the hydrophobic PHA inclusions it creates. Although this feature is actively exploited to generate functionalized biobeads via protein engineering, little is known about the structure of the PHA synthase. Here, the surface topology of Ralstonia eutropha PHA synthase was probed to inform rational protein engineering toward the production of functionalized PHA beads. Surface-exposed residues were detected by conjugating biotin to inclusion-bound PHA synthase and identifying the biotin-conjugated lysine and cysteine residues using peptide fingerprinting analysis. The identified sites (K77, K90, K139, C382, C459, and K518) were investigated as insertion sites for the generation of new protein fusions. Insertions of FLAG epitopes into exposed sites K77, K90, K139, and K518 were tolerated, retaining >65 % of in vivo activity. Sites K90, K139, and K518 were also tested by insertion of the immunoglobulin G (IgG)-binding domain (ZZ), successfully producing PHA inclusions able to bind human IgG in vitro. Although simultaneous insertions of the ZZ domain into two sites was permissive, insertion at all three lysine sites inactivated the synthase. The K90/K139 double ZZ insertion had the optimum IgG-binding capacity of 16 mg IgG/g wet PHA beads and could selectively purify the IgG fraction from human serum. Overall, this study identified surface-exposed flexible regions of the PHA synthase which either tolerate protein/peptide insertions or are critical for protein function. This further elucidates the structure and function of PHA synthase and provides new opportunities for generating functionalized PHA biobeads.
authors.affiliation: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors.forename: David O
authors.initials: DO
authors.lastname: Hooks
authors.forename: Bernd H A
authors.initials: BH
authors.lastname: Rehm
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020118:Cupriavidus necator / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D054813:Polyhydroxyalkanoates / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering
chemical_list: D001426:Bacterial Proteins; D007074:Immunoglobulin G; D054813:Polyhydroxyalkanoates; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase