Testing the transacylation site through mutations to KS surface
residues
To assess the proposed transacylation site, PikKS6 surface residues were
mutated within P1 -P6 -P7 , a triketide synthase
composed of PikMod1, PikMod6, and PikMod7 (Figure
5b).24, 25 In the top solution for the
association of PikACP6 and PikKS6, the PikKS6 surface residues N275,
H281, L315, I319, Q322, D382, and D385 make favorable contacts with
PikACP6, while Q217, S230, Q286, S300, R314, Q330, E379, S387, E392, and
T395 make little or no contact. Each of these residues was individually
mutated to alanine withinP1 -P6 -P7 .34 The
prominent putative interface residues N275 and L315 were also
individually mutated to aspartate and valine, respectively. As a
control, a key residue at the condensation site, T140, was individually
mutated to alanine.7, 8P1 -P6 -P7 and its 20 point mutants were
expressed in Escherichia coli K207-3,35and triketide lactone production was measured by LC/MS after 8 days. The
observed decreases in production are consistent with the predicted
transacylation site.