TY - JOUR

T1 - Catalytic self-acylation of type II polyketide synthase acyl carrier proteins

AU - Hitchman, Timothy S.

AU - Crosby, John

AU - Byrom, Kate J.

AU - Cox, Russell J.

AU - Simpson, Thomas J.

N1 - Funding information: We thank David Hopwood and Peter Revill for the gift of S. coelicolor FAS holo-ACP and their useful comments on the manuscript, Mark Ginty for synthesising acetoacetyl NAC, Stuart Findlow for supplying otc holo-ACP and Chris Dempsey and Richard Sessions for help with the diagram of act ACP. We also acknowledge one of the referees for suggesting the urea denaturation experiments. This work was supported by grants from the Engineering and Physical Sciences Research Council (EPSRC) and the Biotechnology and Biological Sciences Research Council (BBSRC). Studentships from the EPSRC TT8.H.) and School of Chemistrv 3. (K.J.B.) are Y aratefullv , acknowledged. R.J.C. gratefully acknowledges the receipt of a Royal Society research grant.

PY - 1998/1

Y1 - 1998/1

N2 - Background: Aromatic polyketides are synthesised in streptomycetes by the successive condensation of simple carboxylic acids, catalysed by multienzyme complexes - the polyketide synthases (PKSs). Polyketide assembly intermediates are covalently linked as thioesters to the holo-acyl carrier protein (ACP) subunit of these type II PKSs. The ACP is primed for chain elongation by the transfer of malonate from malonyl CoA. Malonylation of fatty acid synthase (FAS) ACPs is catalysed by specific malonyl transferase (MT) enzymes. The type II PKS gene clusters apparently lack genes encoding such MT proteins, however. It has been proposed that the MT subunit of the FAS in streptomycetes catalyses malonylation of both FAS and PKS ACPs in vivo. Results: We demonstrate that type II PKS ACPs catalyse self-malonylation upon incubation with malonyl CoA in vitro. The self-malonylation reaction of the actinorhodin C17S holo-ACP has a K(m) for malonyl CoA of 219 μM and a k(cat) of 0.34 min-1. Complete acylation of the PKS ACPs was observed with malonyl, methylmalonyl and acetoacetyl CoAs. No reaction was observed with acetyl and butyryl CoAs and FAS ACPs did not react with any of the substrates. Recombinant FAS MT from Streptomyces coelicolor did not accelerate the rate of malonylation. Conclusions: The catalytic self-acylation of type II PKS ACPs is an unprecedented reaction. We propose a reaction mechanism in which conserved arginines form a salt bridge with the acyl moiety and sequester it from bulk solvent. This work suggests that the β-ketoacyl synthase, chain length factor and ACP may constitute a truly minimal PKS in vivo.

AB - Background: Aromatic polyketides are synthesised in streptomycetes by the successive condensation of simple carboxylic acids, catalysed by multienzyme complexes - the polyketide synthases (PKSs). Polyketide assembly intermediates are covalently linked as thioesters to the holo-acyl carrier protein (ACP) subunit of these type II PKSs. The ACP is primed for chain elongation by the transfer of malonate from malonyl CoA. Malonylation of fatty acid synthase (FAS) ACPs is catalysed by specific malonyl transferase (MT) enzymes. The type II PKS gene clusters apparently lack genes encoding such MT proteins, however. It has been proposed that the MT subunit of the FAS in streptomycetes catalyses malonylation of both FAS and PKS ACPs in vivo. Results: We demonstrate that type II PKS ACPs catalyse self-malonylation upon incubation with malonyl CoA in vitro. The self-malonylation reaction of the actinorhodin C17S holo-ACP has a K(m) for malonyl CoA of 219 μM and a k(cat) of 0.34 min-1. Complete acylation of the PKS ACPs was observed with malonyl, methylmalonyl and acetoacetyl CoAs. No reaction was observed with acetyl and butyryl CoAs and FAS ACPs did not react with any of the substrates. Recombinant FAS MT from Streptomyces coelicolor did not accelerate the rate of malonylation. Conclusions: The catalytic self-acylation of type II PKS ACPs is an unprecedented reaction. We propose a reaction mechanism in which conserved arginines form a salt bridge with the acyl moiety and sequester it from bulk solvent. This work suggests that the β-ketoacyl synthase, chain length factor and ACP may constitute a truly minimal PKS in vivo.

KW - Acyl carrier protein

KW - Fatty acid synthase

KW - Malonyl transferase

KW - Polyketide synthase

UR - http://www.scopus.com/inward/record.url?scp=0031931702&partnerID=8YFLogxK

U2 - 10.1016/S1074-5521(98)90085-0

DO - 10.1016/S1074-5521(98)90085-0

M3 - Article

AN - SCOPUS:0031931702

VL - 5

SP - 35

EP - 47

JO - Chemistry and Biology

JF - Chemistry and Biology

SN - 1074-5521

IS - 1

ER -