Characterization of a novel acyl carrier protein, RkpF, encoded by an operon involved in capsular polysaccharide biosynthesis in Sinorhizobium meliloti.
Rhizobial capsular polysaccharides (RKPs) play an important role in the development of a nitrogen-fixing symbiosis with the plant host and in Sinorhizobium meliloti AK631 functional rkpABCDEF genes are required for the production of RKPs. After cloning the rkpF gene, we overexpressed and purified the derived protein product (RkpF) in Escherichia coli. Like acyl carrier protein (ACP), the RkpF protein can be labeled in vivo with radioactive beta-alanine added to the growth medium. If homogeneous RkpF protein is incubated with radiolabeled coenzyme A in the presence of purified holo-ACP synthase from E. coli, an in vitro transfer of 4'-phosphopantetheine to the RkpF protein can be observed. The conversion from apo-RkpF protein to holo-RkpF protein seems to go along with a major conformational change of the protein structure, because the holo-RkpF protein runs significantly faster on native polyacrylamide gel electrophoresis than the apo-RkpF protein. Electrospray mass spectrometric analysis reveals a mass of 9,585 for the apo-RkpF protein and a mass of 9,927 for the holo-RkpF protein. Our data show that RkpF is a novel ACP.
Epple
G
G
Institute of Biotechnology, Technical University Berlin, Berlin, Germany.
van der Drift
K M
KM
Thomas-Oates
J E
JE
Geiger
O
O
D000213:Acyl Carrier Protein / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016667:Bacterial Capsules / Q000737:chemistry*; D001426:Bacterial Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009876:Operon; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D012231:Rhizobium / Q000737:chemistry* / Q000235:genetics; D015091:beta-Alanine / Q000378:metabolism
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D015091:beta-Alanine; D003065:Coenzyme A