Journal of the American Chemical Society
Communication
pyrrolyl-S-CalN3 substrate while maintaining reactivity for the
dichlorinated substrate (Figure 3F). Thus, the Met222/
Leu221 side chains participate in the selection for the correct
derivatization state of the pyrrolyl molecular cargoes in KS
active sites. Active-site residues also tailor the substrate
specificities of intermediary KS domains in collinear PKS
pathways.5,6 Overall, our data demonstrate that the strict
substrate selectivity of the very first KS active site in collinear
PKS assembly lines can perform a gatekeeping role to
determine whether polyketide extension can be initiated.
Intermolecular protein−protein interactions underlie poly-
ketide assembly.34 CPs sequester their molecular cargoes; the
structure of pyrrolyl-S-PltL demonstrates that the phospho-
pantetheine arm folds in such a way that the pyrrole ring binds
at a hydrophobic patch on the PltL surface.35 Hence, the
exclusion of pyrrolyl-S-PltL by PltB but acceptance of pyrrolyl-
S-CalN3 by CalA could depend on how PltL and CalN3
differentially present the pyrrolyl substrates to the KS. To
query this hypothesis, we tested pyrrolyl- and dichloropyrrolyl-
S-CalN3 as substrates for PltB. Compared with dichloropyr-
rolyl-S-PltL, the preference for dichloropyrrolyl-S-CalN3 was
reduced. However, pyrrolyl-S-CalN3, just like pyrrolyl-S-PltL,
was not depleted by PltB at all (Figure 4A). This result
compared with PltB (Figure 3A,B). Both PltB and CalA
demonstrated the least preference for HrmL. Phylogenetically,
HrmL is more distant to PltL and CalN3 than any of the other
Here we have demonstrated a gatekeeper role for KSs in
initiating polyketide extension. Combinatorial expansion of the
chemical space explored by pyrrolyl natural products will
require an understanding of the intermolecular interactions
that occur between the molecular cargo, the donor CP, and the
initiating PKS modules. While structural models have guided
modulation of KS substrate selectivities,5,6,37,38 this study
provides evidence that certain KSs, such as CalA, are already
substrate-promiscuous and should be prioritized for further
exploration for diversity-generating efforts.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
Materials and methods for protein expression and
purification, chemical synthesis and spectroscopic
characterization data, assay and data analysis procedures,
and computational details (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Vinayak Agarwal − School of Chemistry and Biochemistry and
School of Biological Sciences, Georgia Institute of Technology,
Authors
Dongqi Yi − School of Chemistry and Biochemistry, Georgia
Institute of Technology, Atlanta, Georgia 30332, United
States
Atanu Acharya − School of Physics, Georgia Institute of
Technology, Atlanta, Georgia 30332, United States;
James C. Gumbart − School of Chemistry and Biochemistry
and School of Physics, Georgia Institute of Technology,
Figure 4. KS activity for noncognate donor CPs. (A) Relative
depletion of dichloropyrrolyl-S-CPs by PltB. Depletion of pyrrolyl-S-
CalN3, the cognate substrate for CalA, is marked by the arrow. (B)
Relative depletion of pyrrolyl-S-CPs by CalA. Depletion of
dichloropyrrolyl-S-PltL, the cognate substrate for PltB, is marked by
the arrow.
Will R. Gutekunst − School of Chemistry and Biochemistry,
Georgia Institute of Technology, Atlanta, Georgia 30332,
United States
demonstrates that while a penalty is indeed paid when the
donor CP for PltB is changed from PltL to CalN3, the
principal determinant for catalysis is the recognition of the
molecular cargo by the KS. CalA depleted both pyrrolyl-S-PltL
and dichloropyrrolyl-S-PltL (Figure 4B). The extents and rates
of depletion of pyrrolyl-S-CalN3 and pyrrolyl-S-PltL by CalA
were similar (Figures 4B and S39).
Complete contact information is available at:
Notes
The authors declare no competing financial interest.
We also tested the ability of the type II NRPS CPs Mpy15,
Clz18, and HrmL to pair with PltB and CalA. Mpy15 and
Clz18 deliver thiotemplated 4,5-dichloropyrrole to PKS
modules in the respective production of marinopyrroles27
and chlorozidine,26 and HrmL delivers 5-chloropyrrole to an
NRPS module for the production of hormaomycin.36 The
activities of PltB and CalA were tested for their cognate
thiotemplated substrates acylated to these different donor CPs
of different donor CPs than CalA. A marked decrease in
acceptance for Mpy15 and Clz18 was observed for CalA
ACKNOWLEDGMENTS
■
The authors acknowledge support from the National Science
Foundation (NSF) (CHE-2004030 to J.C.G. and V.A.), N.
Garg for access to mass spectrometers, and A. Keatinge-Clay
for the gift of MatB. Computational resources were provided
by the Extreme Science and Engineering Discovery Environ-
ment (XSEDE Allocation TG-MCB130173, NSF), and the
Partnership for an Advanced Computing Environment
(PACE) at Georgia Institute of Technology.
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J. Am. Chem. Soc. 2021, 143, 7617−7622