title: Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein.
abstract: 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.
authors.affiliation: School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Arthur
authors.forename: Anna
authors.initials: A
authors.lastname: Szafranska
authors.forename: Simon E
authors.initials: SE
authors.lastname: Evans
authors.forename: Stuart C
authors.initials: SC
authors.lastname: Findlow
authors.forename: Steven G
authors.initials: SG
authors.lastname: Burston
authors.forename: Philip
authors.initials: P
authors.lastname: Owen
authors.forename: Ian
authors.initials: I
authors.lastname: Clark-Lewis
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Simpson
authors.forename: John
authors.initials: J
authors.lastname: Crosby
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Crump
mesh_terms: 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
chemical_list: D000213:Acyl Carrier Protein; D001059:Apoproteins; D008314:Malonates; D048630:Polyketide Synthases; C030290:malonic acid; C087193:polyketide beta-ketoacylsynthase; D051080:Acyl-Carrier Protein S-Malonyltransferase