Cloning, overproduction, and characterization of the Escherichia coli holo-acyl carrier protein synthase.
Holo-acyl carrier protein synthase (ACPS) transfers the 4'-phosphopantetheine (4'-PP) moiety from coenzyme A (CoA) to Ser-36 of acyl carrier protein (ACP) in Escherichia coli. This post-translational modification renders holo-ACP capable of acyl group activation via thioesterification of the cysteamine thiol of 4'-PP. We have purified E. coli ACPS to near homogeneity by exploiting the ability to refold ACPS and reconstitute its activity after elution from an apo-ACP affinity column under denaturing conditions. N-terminal sequencing of ACPS allowed us to identify dpj, an essential gene of previously unknown function, as the structural gene for ACPS. We report herein the 70,000-fold purification of wild-type ACPS and the overproduction and initial characterization of recombinant ACPS from E. coli.
Lambalot
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RH
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Walsh
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CT
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification; D017855:Transferases (Other Substituted Phosphate Groups) / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase