Chem. Biol. 19, 329–339 (2012)

Natural product biosynthesis machineries use a combination of enzyme modules to create a diverse array of natural products. Within polyketide synthases, acyltransferases (ATs) are responsible for loading substrates onto appropriate carrier proteins, with some pathways containing both a 'standard' AT (AT1) and what seems to be a duplicate copy of the AT domain (AT2). A previous observation of premature release of pathway intermediates in a mutant of the bacillaene cluster suggested the pathway contained a proofreading mechanism, but the protein responsible for this function was unknown. In efforts to track down this enzyme, Jensen et al. noted that ATs are functionally related to the putative proofreader. To explore this idea, they focused on the pederin pathway, which contains one AT1 (PedD) and one AT2 (PedC). In vitro tests of both enzymes showed that, in line with normal AT function, PedD could transfer malonyl units onto any of four carrier proteins. PedC, however, was inactive. In contrast, assays testing for possible proofreading activity that showed PedD was inactive, whereas PedC was able to hydrolyze four of six putative substrates. PedC was further tested on carrier proteins modified with hexanoyl, malonyl or acetyl groups and hydrolyzed the hexanoyl and acetyl but not malonyl groups. These combined results indicate that PedC, and most likely other AT2 homologs, determine product integrity by allowing incorporation of only the correct malonyl substrates.