FT-ICR-MS Characterization of Intermediates in the Biosynthesis of the α-Methylbutyrate Side Chain of Lovastatin by the 277 kDa Polyketide Synthase LovF

摘要

There are very few fungal polyketide synthases that have been characterized bymass spectrometry.nIn this paper we describe the in vitro reconstitution and FT-ICR-MS verification of the full activity of an intactn277 kDa fungal polyketide synthase LovF of the lovastatin biosynthetic pathway. We report here both thenverification of the reconstitution of fully functional holo-LovF by using 13nC-labeled malonyl-CoA to formnR-methylbutyrate functionality and also detection of five predicted intermediates covalently bound to then40n-phosphopantetheine at the acyl carrier protein (ACP) active site utilizing the phosphopantetheine ejectionnassay and high-resolution mass spectrometry.Under in vitro conditions, the diketide acetoacetyl intermediatendid not accumulate on the ACP active site of holo-LovF following incubation with malonyl-CoA substrate.nWe found that incubation of holo-LovF with acetoacetyl-CoA served as an effective means of loading thendiketide intermediate onto the ACP active site of LovF. Our results demonstrate that subsequent R-methyl-nation of the acetoacetyl intermediate stabilizes the intermediate onto the ACP active site and facilitatesnthe formation and mass spectrometric detection of additional intermediates en route to the formation ofnR-methylbutyrate.