INTRODUCTION
Modular polyketide synthases (PKS’s) are multidomain, enzymatic assembly
lines that play the major role in the production of important
pharmaceuticals, such as the antibiotic erythromycin and the anticancer
agent epothilone.1-3 Each module is minimally
comprised of an acyl carrier protein (ACP) domain that shuttles
polyketide intermediates between enzymatic domains using a
phosphopantetheinyl arm and a ketosynthase (KS) domain that selects
which intermediates to extend and pass along the assembly line (Figure
1). A module may also contain an acyltransferase (AT) domain that
selects α-carboxyacyl extender units, such as a malonyl or methylmalonyl
group, and processing domains, such as a ketoreductase (KR), dehydratase
(DH), or enoylreductase (ER), that perform chemistry on extended
intermediates. While the high-resolution structures of each of these
domains have been known for many years, the interfaces between them are
only now being characterized.4
Each KS of an assembly line is thought to possess distinct docking sites
for the upstream and downstream ACP’s with which it
collaborates.5, 6 It was hypothesized that the
upstream ACP docks at a transacylation site to present intermediates for
transfer to the reactive KS cysteine and the downstream ACP docks at an
extension site to collect the intermediate through a decarboxylative
Claisen-like condensation with a bound α-carboxyacyl extender unit. The
extension site has been revealed by cryogenic-electron microscopy
(cryo-EM) and x-ray crystallography.7-9 In
cryo-EM studies of both PikAIII (the third polypeptide of the Pikromycin
synthase)10 and Mycobacterium smegmatisPks13 (PDB: 8CUY, 8CV1),11 other ACP/KS
interactions were observed; however, in each of these interactions the
phosphopantetheinylated serine of ACP is more than 28 Å from the KS
reactive cysteine. As the thioester carbon attacked by the KS reactive
cysteine can only stretch 19.2 Å from the phosphopantetheinylated serine
oxygen, none of these interfaces represent the transacylation site.
Improvements in folding/docking algorithms are enabling the reliable
prediction of physiologically relevant domain-domain
interfaces.12-15 Since ACP’s and downstream
KS’s collaborate within the PKS module (updated boundary
used16, 17 ) to gatekeep for processed
polyketide intermediates,18 evolutionarily
co-migrate,16, 17, 19, 20 and are best
maintained together within engineered assembly
lines,21-24 we hypothesized that modern
folding/docking algorithms would detect the interface between these
domains.
The AlphaFold-Multimer14 predictions for 50
natural ACP/KS pairs from diverse, well-characterized PKS’s are highly
similar.2 Half of the top solutions contain a
conformational change of the KS TxLGDP motif at the putative docking
interface that enables a closer approach (17.2 Å vs. 20.9 Å, on average)
of the phosphopantetheinylated ACP serine and the reactive KS cysteine.
These solutions provide a molecular description of how acyl-ACP’s dock
with KS’s to enable the transacylation reaction. A model triketide
lactone synthase composed of the 1st,
6th, and 7th modules of the
Pikromycin synthase, P1 -P6 -P7 , was employed
to test the predicted transacylation site.24,
25 The relative decreases in the activities of 20 variants containing
mutations to surface residues on the KS from the 6thmodule, PikKS6, are consistent with the proposed interface. The data
suggest that two residues conserved on the surface of KS, an asparagine
in position 275 and a leucine in position 315, play major roles in
binding acyl-ACP’s for transacylation.