Purification and characterization of acyl carrier protein from two cyanobacteria species.
The acyl carrier protein (ACP), an essential protein cofactor for fatty acid synthesis, has been isolated from two cyanobacteria: the filamentous, heterocystous, Anabaena variabilis (ATCC 29211) and the unicellular Synechocystis 6803 (ATCC 27184). Both ACPs have been purified to homogeneity utilizing a three-column procedure. Synechocystis 6803 ACP was purified 1800-fold with 67% yield, while A. variabilis ACP was purified 1040-fold with 50% yield. Yields of 13.0 micrograms ACP/g Synechocystis 6803 and 9.0 micrograms ACP/g A. variabilis were achieved. Amino acid analysis indicated that these ACPs were highly charged acidic proteins similar to other known ACPs. Sequence analysis revealed that both cyanobacterial ACPs were highly conserved with both spinach and Escherichia coli ACP at the phosphopantetheine prosthetic group region. Examining the probability of alpha-helix and beta-turn regions in various ACPs, showed that cyanobacterial ACPs were more closely related to E. coli ACP than spinach ACP I. Immunoblot analysis and a competitive binding assay for ACP illustrated that both ACPs bound poorly to spinach ACP I antibody. SDS/PAGE and native PAGE of Synechocystis 6803 ACP and A. variabilis ACP showed that cyanobacteria ACPs co-migrated with E. coli ACP and had relative molecular masses of 18,100 and 17,900 respectively. Both native and urea gel analysis of acyl-ACP products from fatty acid synthase reactions demonstrated that bacterial ACPs and plant ACP gave essentially the same metabolic products when assayed using either bacterial or plant fatty acid synthase. A. variabilis and Synechocystis 6803 ACP could be acylated using E. coli acyl ACP synthetase.
Froehlich
J E
JE
Department of Chemistry, Miami University, Oxford, OH 45056.
Poorman
R
R
Reardon
E
E
Barnum
S R
SR
Jaworski
J G
JG
D000213:Acyl Carrier Protein / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D000458:Cyanobacteria / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid
D000213:Acyl Carrier Protein; D010169:Palmitic Acids; D019308:Palmitic Acid; D004247:DNA