Ketosynthases
(KSs) catalyze carbon–carbon bond-forming
reactions in fatty acid synthases (FASs) and polyketide synthases
(PKSs). KSs utilize a two-step ping-pong kinetic mechanism to carry
out an overall decarboxylative thio-Claisen condensation that can
be separated into the transacylation and condensation reactions. In
both steps, an acyl carrier protein (ACP) delivers thioester-tethered
substrates to the active sites of KSs. Therefore, protein–protein
interactions (PPIs) and KS-mediated substrate recognition events are
required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF
and FabB, in complex with E. coli ACP,
AcpP, revealed distinct conformational states of two active site KS
loops. These loops were proposed to operate via a gating mechanism
to coordinate substrate recognition and delivery followed by catalysis.
Here, we interrogate this proposed gating mechanism by solving two
additional high-resolution structures of substrate-engaged AcpP–FabF
complexes, one of which provides the missing AcpP–FabF gate-closed
conformation. Clearly defined interactions of one of these active
site loops with AcpP are present in both the open and closed conformations,
suggesting AcpP binding triggers or stabilizes gating transitions,
further implicating PPIs in carrier protein-dependent catalysis. We
functionally demonstrate the importance of gating in the overall KS
condensation reaction and provide experimental evidence for its role
in the transacylation reaction. Furthermore, we evaluate the catalytic
importance of these loops using alanine-scanning mutagenesis and also
investigate chimeric FabF constructs carrying elements found in type
I PKS KS domains. These findings broaden our understanding of the
KS mechanism, which advances future engineering efforts in both FASs
and evolutionarily related PKSs.