Gatekeeping Ketosynthases Dictate Initiation of Assembly Line Biosynthesis of Pyrrolic Polyketides

Article abstract of DOI:10.1021/jacs.1c02371

Assembly line biosynthesis of polyketide natural products involves checkpoints where identities of thiotemplated intermediates are verified before polyketide extension reactions are allowed to proceed. Determining what these checkpoints are and how they operate is critical for reprogramming polyketide assembly lines. Here we demonstrate that ketosynthase (KS) domains can perform this gatekeeping role. By comparing the substrate specificities for polyketide synthases that extend pyrrolyl and halogenated pyrrolyl substrates, we find that KS domains that need to differentiate between these two substrates exercise high selectivity. We additionally find that amino acid residues in the KS active site facilitate this selectivity and that these residues are amenable to rational engineering. On the other hand, KS domains that do not need to make selectivity decisions in their native physiological context are substrate-promiscuous. We also provide evidence that delivery of substrates to polyketide synthases by non-native carrier proteins is accompanied by reduced biosynthetic efficiency.

Full text of DOI:10.1021/jacs.1c02371

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Gatekeeping Ketosynthases Dictate Initiation of Assembly Line
Biosynthesis of Pyrrolic Polyketides
Dongqi Yi, Atanu Acharya, James C. Gumbart, Will R. Gutekunst, and Vinayak Agarwal*
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ABSTRACT: Assembly line biosynthesis of polyketide natural products involves checkpoints where identities of thiotemplated
intermediates are verified before polyketide extension reactions are allowed to proceed. Determining what these checkpoints are and
how they operate is critical for reprogramming polyketide assembly lines. Here we demonstrate that ketosynthase (KS) domains can
perform this gatekeeping role. By comparing the substrate specificities for polyketide synthases that extend pyrrolyl and halogenated
pyrrolyl substrates, we find that KS domains that need to differentiate between these two substrates exercise high selectivity. We
additionally find that amino acid residues in the KS active site facilitate this selectivity and that these residues are amenable to
rational engineering. On the other hand, KS domains that do not need to make selectivity decisions in their native physiological
context are substrate-promiscuous. We also provide evidence that delivery of substrates to polyketide synthases by non-native carrier
proteins is accompanied by reduced biosynthetic efficiency.
ype I polyketide synthases (PKSs) are multimodular
enzymes that construct polyketide natural products.1 The
the PltB CP by the AT enables the KS to catalyze the
decarboxylative chain extension. Additional polyketide exten-
sions followed by aromatization afford 1 (Figure S1). The
precursor to 4,5-dichloropyrrolyl-S-PltL, pyrrolyl-S-PltL,12 is
also available in situ to the PltB PKS module (Figure 1B).
However, deschloro-1 is not produced (Figure S2). This is in
contrast to the biosynthetic scheme for calcimycin (2) (Figure
1C), in which pyrrole (pyrrolyl-S-CalN3) acts as the
physiological substrate to initiate polyketide extension by
CalA.13
Why is pyrrolyl-S-PltL rejected as a substrate by PltB while
pyrrolyl-S-CalN3 is accepted by CalA? To query the specificity
determinants for PltB and CalA activities, we adopted an
approach comparing the intermolecular recognition events that
dictate polyketide extension of pyrrolic molecular cargoes.
First, we determined the KS−AT−CP module boundaries for
the bimodular PltB and CalA proteins by alignment with other
KS−AT and CP crystal structures.14−16 The tridomain
sequences were expressed in Escherichia coli, and the PltB
and CalA KS−AT−CP modules were purified (Figure S3).
The CPs PltL and CalN3 were purified in their apo forms.
A library of pyrrolyl-S-pantetheines were synthesized
(Scheme 1). The previously described synthesis of S-acyl
pantetheines involved thioesterification of the acyl groups to
give acetonide-protected pantetheine and subsequent acid
deprotection to unmask the 1,3-diol.17,18 However, we found
the halogenated pyrrole derivatives to be labile toward acidic
T
assembly line biosynthesis of polyketides involves repetitive
steps: choice of an extender unit by the acyltransferase (AT)
domain, decarboxylative Claisen condensation of this extender
unit to the polyketide by the ketosynthase (KS) domain, and
reductive tailoring of the β-carbonyl by ketoreductase,
dehydratase, and enoyl reductase domains. Substrates for
these PKS domains are thioesterified to carrier proteins (CPs).
The ATs and the reductive tailoring domains were traditionally
thought to determine the diversity of polyketide natural
products.2 The role of KSs in determining the polyketide
diversity is relatively less well studied. Intermediary KSs in
collinear PKS assembly lines are evolutionarily linked to the
CP and the tailoring domains that precede them,1,3,4 and as
polyketide extension progresses, the selectivity of these
intermediary KSs can constrain the extension of noncognate
substrates.5−8 Here we demonstrate that the strict selectivity of
the very first KS in a collinear PKS assembly line can perform a
gatekeeping function by precluding the initiation of polyketide
extension of noncognate substrates and that the selectivity and
promiscuity of initiating KSs are modulated in tune with the
physiological natural product biosynthetic scheme.
The biosynthesis of several pyrrole-containing natural
products involves polyketide extensions. Thioesterified pyr-
roles are delivered by a type II nonribosomal peptide
synthetase (NRPS) adenylation−oxidation cascade that
oxidizes the L-proline pyrrolidine to pyrrole (Figure 1A).9−11
Thiotemplated pyrroles can be modified, as exemplified by
dichlorination in the biosynthesis of pyoluteorin (1) (Figure
1B).12 The thiotemplated dichloropyrrole is then handed off,
without intermediary offloading, from the type II NRPS donor
CP PltL to the type I PKS PltB. This handoff occurs by
transthioesterification of the molecular cargo from the donor
CP to the active-site cysteine thiol of the KS. Malonylation of
Received: March 2, 2021
Published: May 14, 2021
https://doi.org/10.1021/jacs.1c02371
J. Am. Chem. Soc. 2021, 143, 7617−7622
© 2021 American Chemical Society
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Figure 1. Biosynthesis of pyrrolic polyketides. (A) A type II NRPS furnishes the pyrrolyl-S-CPs. (B) Halogenation of pyrrolyl-S-PltL and
polyketide elongation by PKS PltB (Cys183 is the KS active-site residue). (C) Polyketide extension by CalA.
were extended to the corresponding S-acyl CoAs, followed by
enzymatic transfer of the S-acyl phosphopantetheines to the
donor CPs (Figures S18−S34).17,18,20
Scheme 1. Synthesis of S-Acyl Pantetheines 5−12
With the substrates in hand, we compared the abilities of the
PltB and CalA KS−AT−CP modules to extend the pyrrolic
cargoes. Malonyl-CoA required for the malonylation of the
PltB CP by the AT was exogenously provided. For CalA,
methylmalonyl-CoA was generated in situ by the CoA ligase
MatB (Figure S35).21 PltB Cys183Ala and CalA Cys182Ala
were used to establish negative controls.22 Upon incubation of
PltB with a 2-fold molar excess of dichloropyrrolyl-S-PltL and
excess malonyl-CoA, we detected the formation of a diketide
product; this diketide was not observed with the PltB
Cys183Ala enzyme (Figure S36). Dichloropyrrolyl-S-PltL was
completely consumed; no consumption of the substrate was
observed for the PltB Cys183Ala enzyme (Figure S37).
The abundance of the diketide detected was not
reproducible. Thus, we relied on quantifying the abundance
of the leftover S-acyl donor CP as a proxy for the extent of the
reaction. Compared with complete consumption of dichlor-
opyrrolyl-S-PltL, the consumption of pyrrolyl-S-PltL was
negligible (Figure 2A). Physiologically, this implies that despite
its presence in the molecular milieu within the producer of 1,
pyrrolyl-S-PltL is not accepted as a substrate by the PltB KS,
thus precluding the production of deschloro-1.
We tested the activity of PltB for other pyrrolyl derivatives.
Both 4- and 5-chloropyrrolyl-S-PltL were accepted as
substrates, but with a reduced preference relative to 4,5-
dichloropyrrole (Figure 2A). Bromine could replace chlorine;
4,5-dibromopyrrolyl-S-PltL was accepted well. However, the
activity was diminished for the 3,4,5-tribromo derivative.
Methyls could not replace halides. Methylpyrroles are observed
in aminocoumarin natural products,23 where 5-methylpyrrole
does not undergo polyketide extension but is instead esterified
to afford glycosyl moieties. We observed a marked decrease in
preference for 5-methylpyrrole. An even further reduction in
deprotection. Instead, D-pantetheine (4) was accessed by
disulfide reduction of D-pantethine (3) and used directly for
ligation to pyrrolyl-2-carboxylic acids to furnish 5−11 (Figures
S4−S17). Molecule 12 was accessed by tribromination of the
pyrrolyl methyl ester followed by conversion to an acyl
chloride, which was thioesterified to 4.19 Compounds 5−12
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Figure 2. KS specificities. (A) Depletion of the end-point substrate S-
acyl CP by PltB and CalA. (B, C) Time-dependent depletion of all
substrates that demonstrated full consumption in the end-point assay
by (B) CalA and (C) PltB.
preference was observed for 4,5-dimethylpyrrolyl-S-PltL
(Figure 2A). Overall, we posit that substitutions on the
pyrrole ring have a strong influence on the PltB KS activity.
Compared with PltB, the CalA KS demonstrated relaxed
substrate selectivity. Complete consumption of pyrrolyl- and
dichloropyrrolyl-S-CalN3 substrates along with other halo-
genated derivatives was observed (Figure 2A). As before, a
reduction in substrate consumption was observed when the 3-
position was brominated. Methyl substituents were less
preferred than the halides; however, the distinction was less
pronounced compared with PltB (Figure 2A). Querying time-
dependent substrate consumption for all of the substrates that
were fully depleted by CalA revealed that the rate of
consumption of the physiological substrate pyrrole-S-CalN3
was lower than that of dichloropyrrolyl-S-CalN3 (Figure 2B).
The faster consumption of dichloropyrrolyl-S-CalN3 was not
due to substrate degradation (Figure S37). These data allow us
to posit that the PltB KS exercises substrate selectivity, as it
needs to differentiate between thiotemplated cargoes within
the biosynthetic milieu. On the other hand, the CalA KS,
which does not encounter differentially modified pyrrolic
substrates, has relaxed substrate selectivity. Without an
evolutionary pressure to maintain fidelity, relaxation in
substrate selectivity is observed for primary and secondary
metabolic enzymes.24 Dichloro- and dibromopyrrole substrates
were consumed by CalA and PltB at similar rates (Figure
2B,C).
Figure 3. KS active site. (A) KS sequence alignment. Sequences for
(from top to bottom) 1, armeniaspirol,25 chlorizidine,26 marinopyr-
role,27 pyrrolomycin,28 pyralomycin,29 pyrronazol B,30 2, and
DKxanthene31 KSs are illustrated. (B) The PltB KS active site. The
Cys183-His318-His355 catalytic triad22 side chains are illustrated.
(C) ESPs mapped onto the electron densities (isovalue = 0.0004) for
(top) dichloropyrrolyl- and (bottom) dibromopyrrolyl-S-N-acetylcys-
teamines. Regions of negative potential and positive potential (>0.01
au) are displayed in red and blue, respectively. ESPs between 0.00 and
0.01 are displayed with a color gradient from red to blue. (D)
Substrate specificity of the PltB Met222Leu mutant compared with
the wild type. (E, F) Time-dependent consumption of pyrrolyl- and
dichloropyrrolyl substrates by wild-type and mutant PltB (Met222-
Leu) and CalA (Leu221Met).
maps for dichloro- and dibromopyrrole-N-acetylcysteamines
computed at the wb97X-D/aug-cc-pVTZ level of theory
demonstrated the presence of complementary halogen-
bonding σ holes on the halogen substituents on the pyrrole
rings (Figures 3C and S38). To probe the role of Met222 in
determining the KS specificity, the substrate selectivity of the
PltB Met222Leu enzyme was queried. The PltB Met222Leu
mutant demonstrated a 4-fold increase in ability to deplete the
pyrrolyl-S-PltL substrate while maintaining activity for
dichloropyrrolyl-S-PltL (Figure 3D,E). Consumption of 4-
and 5-chloropyrrolyl-S-PltL was also enhanced, mirroring the
activity of CalA. Introducing the complementary mutation
Leu221Met in CalA led to a reduction in depletion of the
Sequence alignment identified a methionine residue that was
conserved for all KSs that extend dichloropyrroles (PltB
Met222; Figure 3A). Homology structure of the PltB KS-AT
didomain places the Met222 side chain within the KS active
site (Figure 3B). The methionine thioether is a validated
halogen-bonding partner.32,33 Electrostatic potential (ESP)
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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
type II NRPS CPs tested (Figure S46).
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
https://pubs.acs.org/doi/10.1021/jacs.1c02371.
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,
Atlanta, Georgia 30332, United States; orcid.org/0000-
0002-2517-589X; Email: vagarwal@gatech.edu
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;
orcid.org/0000-0002-6960-7789
James C. Gumbart − School of Chemistry and Biochemistry
and School of Physics, Georgia Institute of Technology,
Atlanta, Georgia 30332, United States; orcid.org/0000-
0002-1510-7842
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:
https://pubs.acs.org/10.1021/jacs.1c02371
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
(Figure S40−45). We observed that PltB was more accepting
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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