Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein.
During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.
Arthur
Christopher J
CJ
School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK.
Szafranska
Anna
A
Evans
Simon E
SE
Findlow
Stuart C
SC
Burston
Steven G
SG
Owen
Philip
P
Clark-Lewis
Ian
I
Simpson
Thomas J
TJ
Crosby
John
J
Crump
Matthew P
MP
D000213:Acyl Carrier Protein / Q000138:chemical synthesis* / Q000378:metabolism*; D051080:Acyl-Carrier Protein S-Malonyltransferase / Q000378:metabolism; D001059:Apoproteins / Q000138:chemical synthesis / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D008314:Malonates / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D048630:Polyketide Synthases / Q000378:metabolism*; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization; D048372:Streptomyces coelicolor / Q000201:enzymology
D000213:Acyl Carrier Protein; D001059:Apoproteins; D008314:Malonates; D048630:Polyketide Synthases; C030290:malonic acid; C087193:polyketide beta-ketoacylsynthase; D051080:Acyl-Carrier Protein S-Malonyltransferase