The recombinant spinach acyl-acyl carrier protein-I expressed in Escherichia coli is the 18:1 delta 11(cis) thioester.
A synthetic spinach acyl carrier protein-I (ACP-I) gene was cloned and expressed in the Escherichia coli beta-alanine auxotroph SJ16 (P. D. Beremand et al. (1987) Arch. Biochem. Biophys. 256, 90-100). After characterization of the transformed cells and purification of the protein product it was evident that 50% of the recombinant spinach ACP-I was acylated during early log-phase growth (D. J. Guerra et al. (1988) J. Biol. Chem. 263, 4386-4391). We have purified the recombinant acyl-acyl carrier protein-I to greater than 90% homogeneity and have made a fatty acid methyl ester of the delipidated and trypsin-treated preparation. We have found that the acyl moiety attached to recombinant spinach acyl carrier protein-I is 18:1 delta 11(cis) (cis-vaccenic acid) a major unsaturated end product of Escherichia coli de novo fatty acid synthesis. This result reflects previous work (D. S. Guerra et al. (1986) Plant Physiol. 82, 448-453) which suggested the acyl carrier protein-I structure has evolved from ancestral ACP structures to accommodate the eukaryotic pathway of lipid synthesis in higher plants. The accumulation of recombinant 18:1 delta 11(cis) acyl carrier protein-I in transformed E. coli SJ16 cells attests to the poor reactivity of this substrate to acyl transferase reactions and may help explain the lack of effect on pools of fatty acids found in vivo.
Guerra
D J
DJ
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
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D000213:Acyl Carrier Protein / Q000096:biosynthesis* / Q000302:isolation & purification; D000215:Acylation; D002845:Chromatography / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D005822:Genetic Vectors; D006868:Hydrolysis; D010170:Palmitoyl-CoA Hydrolase / Q000378:metabolism; D010945:Plants, Edible / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D014357:Trypsin
D000213:Acyl Carrier Protein; D011994:Recombinant Proteins; D010170:Palmitoyl-CoA Hydrolase; D014357:Trypsin