Capturing transient biosynthetic intermediates on the post-translationally modified polyketide synthase involved in the biosynthesis of lovastatin by FT-ICR-MS.

摘要

We were able to directly detect covalently bound biosynthetic intermediates from a completely intact and functional fungal polyketide systhase. Using a variety of substrates, it was possible to detect all but one of the 7 chemical species predicted to be found covalently linked to the ACP active site. Utilizing the phosphopantetheine ejection assay (PEA) it was possible to differentiate between ejection ions with masses that differed by less than 0.1 Da. At steady-state conditions, the diketide (3) was not present on the ACP of LovF, and most likely it resided at the ketosynthase domain active site until it could pass quickly through the subsequent tailoring steps. We were able to detect that LovF utilized ~(13)C_(3)-malonyl-CoA and acetoacetyl-CoA as a substrate for the additional methylation, reductions, and dehydration reactions to yield alpha-methylbutyryl-S-LovF.