Mutagenesis supports AlphaFold prediction of how modular polyketide
synthase acyl carrier proteins dock with downstream ketosynthases
Abstract
The docking of an acyl carrier protein (ACP) domain with a downstream
ketosynthase (KS) domain in each module of a polyketide synthase (PKS)
helps ensure accurate biosynthesis. If the polyketide chain bound to the
ACP has been properly modified by upstream processing enzymes and is
compatible with gatekeeping residues in the KS tunnel, a transacylation
reaction can transfer it from the 18.1-Å phosphopanthetheinyl arm of the
ACP to the reactive cysteine of the KS. AlphaFold-Multimer predicts a
general interface for these transacylation checkpoints. Half of the
solutions obtained for 50 ACP/KS pairs show the KS motif TxLGDP forming
the first turn of an a-helix, as in reported structures, while half show
it forming a type I b-turn not previously observed. Solutions with the
latter conformation may represent how these domains are relatively
positioned during the transacylation reaction, as the entrance to the KS
active site is relatively open and the phosphopantetheinylated ACP
serine and the reactive KS cysteine are relatively closer - 17.2 Å vs.
20.9 Å, on average. To probe the predicted interface, 20 mutations were
made to KS surface residues within the model triketide lactone synthase
P1- P6- P7. The activities of these mutants
are consistent with the proposed interface.