Ability of Streptomyces spp. acyl carrier proteins and coenzyme A analogs to serve as substrates in vitro for E. coli holo-ACP synthase.
BACKGROUND
The polyketide natural products are assembled by a series of decarboxylation/condensation reactions of simple carboxylic acids catalyzed by polyketide synthase (PKS) complexes. The growing chain is assembled on acyl carrier protein (ACP), an essential component of the PKS. ACP requires posttranslational modification on a conserved serine residue by covalent attachment of a 4'-phosphopantetheine (P-pant) cofactor to yield active holo-ACP. When ACPs of Streptomyces type II aromatic PKS are overproduced in E. coli, however, typically little or no active holo-ACP is produced, and the ACP remains in the inactive apo-form.


RESULTS
We demonstrate that E. coli holo-ACP synthase (ACPS), a fatty acid biosynthesis enzyme, can catalyze P-pant transfer in vitro to the Streptomyces PKS ACPs required for the biosynthesis of the polyketide antibiotics granaticin, frenolicin, oxytetracycline and tetracenomycin. The catalytic efficiency of this P-pant transfer reaction correlates with the overall negative charge of the ACP substrate. Several coenzyme A analogs, modified in the P-pant portion of the molecule, are likewise able to serve as substrates in vitro for ACPS.


CONCLUSIONS
E coli ACPS can serve as a useful reagent for the preparation of holo-forms of Streptomyces ACPs as well as holo-ACPs with altered phosphopantetheine moieties. Such modified ACPs should prove useful for studying the role of particular ACPs and the phosphopantetheine cofactor in the subsequent reactions of polyketide and fatty acid biosynthesis.
Gehring
A M
AM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Lambalot
R H
RH
Vogel
K W
KW
Drueckhammer
D G
DG
Walsh
C T
CT
D000213:Acyl Carrier Protein / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000378:metabolism; D010204:Pantetheine / Q000031:analogs & derivatives / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013302:Streptomyces / Q000378:metabolism*; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups) / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D000213:Acyl Carrier Protein; D000900:Anti-Bacterial Agents; D017931:DNA Primers; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D010204:Pantetheine; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; C003129:4'-phosphopantetheine; D003065:Coenzyme A