MCAT is not required for in vitro polyketide synthesis in a minimal actinorhodin polyketide synthase from Streptomyces coelicolor.
BACKGROUND
It has been proposed that Streptomyces malonyl CoA: holo acyl carrier protein transacylases (MCATs) provide a link between fatty acid and polyketide biosynthesis. Two recent studies have provided evidence that the presence of MCAT is essential for polyketide synthesis to proceed in reconstituted minimal polyketide synthases (PKSs). In contrast to this, we previously showed that the holo acyl carrier proteins (ACPs) from type II PKSs are capable of catalytic self-malonylation in the presence of malonyl CoA, which suggests that MCAT might not be necessary for polyketide biosynthesis.


RESULTS
We reconstituted a homologous actinorhodin (act) type II minimal PKS in vitro. When act holo-ACP is present in limiting concentrations, MCAT is required by the synthase complex in order for polyketide biosynthesis to proceed. When holo-ACP is present in excess, however, efficient polyketide synthesis proceeds without MCAT. The rate of polyketide production increases with holo-ACP concentration, but at low ACP concentration or equimolar AC:KS:CLF (KS, ketosynthase; CLF, chain length determining factor) concentrations this rate is significantly lower than expected, indicating that free holo-ACP is sequestered by the KS/CLF complex.


CONCLUSIONS
The rate of polyketide biosynthesis is dictated by the ratio of holo-ACP to KS and CLF, as well as by the total protein concentration. There is no absolute requirement for MCAT in polyketide biosynthesis in vitro, although the role of MCAT during polyketide synthesis in vivo remains an open question. MCAT might be responsible for the rate enhancement of malonyl transfer at very low free holo-ACP concentrations or it could be required to catalyse the transfer of malonyl groups from malonyl CoA to sequestered holo-ACP.
Matharu
A L
AL
School of Chemistry University of Bristol Cantock's Close Bristol BS8 1TS UK.
Cox
R J
RJ
Crosby
J
J
Byrom
K J
KJ
Simpson
T J
TJ
D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D007700:Kinetics; D008314:Malonates / Q000378:metabolism; D008316:Malonyl Coenzyme A / Q000378:metabolism; D048630:Polyketide Synthases; D013302:Streptomyces / Q000201:enzymology*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008314:Malonates; D008316:Malonyl Coenzyme A; D048630:Polyketide Synthases; C030290:malonic acid; D000217:Acyltransferases; C087193:polyketide beta-ketoacylsynthase; D051080:Acyl-Carrier Protein S-Malonyltransferase; C494968:fabD protein, E coli; D054889:Fatty Acid Synthase, Type II