Identification of the region of a 14-kilodalton protein of Rhodococcus ruber that is responsible for the binding of this phasin to polyhydroxyalkanoic acid granules.
The function of the polyhydroxyalkanoic acid (PHA) granule-associated GA14 protein of Rhodococcus ruber was investigated in Escherichia coli XL1-Blue, which coexpressed this protein with the polyhydroxybutyric acid (PHB) biosynthesis operon of Alcaligenes eutrophus. The GA14 protein had no influence on the biosynthesis rate of PHB in E. coli XL1-Blue(pSKCO7), but this recombinant E. coli strain formed smaller PHB granules than were formed by an E. coli strain that expressed only the PHB operon. Immunoelectron microscopy with GA14-specific antibodies demonstrated the binding of GA14 protein to these mini granules. In a previous study, two hydrophobic domains close to the C terminus of the GA14 protein were analyzed, and a working hypothesis that suggested an anchoring of the GA14 protein in the phospholipid monolayer surrounding the PHA granule core by these hydrophobic domains was developed (U. Pieper-Fürst, M. H. Madkour, F. Mayer, and A. Steinbüchel, J. Bacteriol. 176:4328-4337, 1994). This hypothesis was confirmed by the construction of C-terminally truncated variants of the GA14 protein lacking the second or both hydrophobic domains and by the demonstration of their inability to bind to PHB granules. Further confirmation of the hypothesis was obtained by the construction of a fusion protein composed of the acetaldehyde dehydrogenase II of A. eutrophus and the C terminus of the GA14 protein containing both hydrophobic domains and by its affinity to native and artificial PHB granules.
Pieper-Fürst
U
U
Institut für Mikrobiologie, Georg-August-Universität Göttingen, Germany.
Madkour
M H
MH
Mayer
F
F
Steinbüchel
A
A
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism; D037102:Lectins / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D037121:Plant Lectins; D011091:Polyesters / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012240:Rhodococcus / Q000378:metabolism* / Q000648:ultrastructure; D017384:Sequence Deletion; D014631:Valerates / Q000378:metabolism; D000094122:Polyhydroxybutyrates
D000217:Acyltransferases; D000445:Aldehyde Oxidoreductases; D001426:Bacterial Proteins; D006885:Hydroxybutyrates; D037102:Lectins; D008565:Membrane Proteins; D010743:Phospholipids; D037121:Plant Lectins; D011091:Polyesters; D011994:Recombinant Proteins; D014631:Valerates; C088136:GA14 protein, Rhodococcus ruber; C008825:phasin; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C073314:poly(3-hydroxyvalerate); C018699:aldehyde dehydrogenase (NAD(P)+); C077225:poly(3-hydroxyalkanoic acid) synthase