Dissecting modular synthases through inhibition: A complementary chemical and genetic approach

Article abstract of DOI:10.1016/j.bmcl.2019.126820

Modular synthases, such as fatty acid, polyketide, and non-ribosomal peptide synthases (NRPSs), are sophisticated machineries essential in both primary and secondary metabolism. Various techniques have been developed to understand their genetic background and enzymatic abilities. However, uncovering the actual biosynthetic pathways remains challenging. Herein, we demonstrate a pipeline to study an assembly line synthase by interrogating the enzymatic function of each individual enzymatic domain of BpsA, a NRPS that produces the blue 3,3′-bipyridyl pigment indigoidine. Specific inhibitors for each biosynthetic domain of BpsA were obtained or synthesized, and the enzymatic performance of BpsA upon addition of each inhibitor was monitored by pigment development in vitro and in living bacteria. The results were verified using genetic mutants to inactivate each domain. Finally, the results complemented the currently proposed biosynthetic pathway of BpsA.

Full text of DOI:10.1016/j.bmcl.2019.126820

Journal Pre-proofs
Dissecting modular synthases through inhibition: a complementary chemical
and genetic approach
Christopher R. Vickery, Ian P. McCulloch, Eva C. Sonnenschein, Joris Beld,
Joseph Noel, Michael D. Burkart
PII:
S0960-894X(19)30789-9
DOI:
https://doi.org/10.1016/j.bmcl.2019.126820
BMCL 126820
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
29 August 2019
8 November 2019
9 November 2019
Please cite this article as: Vickery, C.R., McCulloch, I.P., Sonnenschein, E.C., Beld, J., Noel, J., Burkart, M.D.,
Dissecting modular synthases through inhibition: a complementary chemical and genetic approach, Bioorganic &
Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126820
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Dissecting modular synthases through inhibition: a complementary chemical and
genetic approach
Christopher R. Vickery a b †, Ian P. McCulloch a †, Eva C. Sonnenschein a †, Joris Beld a †, Joseph Noel b and
Michael D. Burkart* a
a Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA.
b Howard Hughes Medical Institute, The Salk Institute for Biological Studies, Jack H. Skirball Center for Chemical Biology and Proteomics, 10010 N. Torrey
Pines Road, La Jolla, CA 92037, USA.
† Authors contributed equally.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Modular synthases, such as fatty acid, polyketide, and non-ribosomal peptide synthases
(NRPSs), are sophisticated machineries essential in both primary and secondary metabolism.
Various techniques have been developed to understand their genetic background and enzymatic
abilities. However, uncovering the actual biosynthetic pathways remains challenging. Herein, we
demonstrate a pipeline to study an assembly line synthase by interrogating the enzymatic
function of each individual enzymatic domain of BpsA, a NRPS that produces the blue 3,3’-
bipyridyl pigment indigoidine. Specific inhibitors for each biosynthetic domain of BpsA were
obtained or synthesized, and the enzymatic performance of BpsA upon addition of each inhibitor
was monitored by pigment development in vitro and in living bacteria. The results were verified
using genetic mutants to inactivate each domain. Finally, the results complemented the currently
proposed biosynthetic pathway of BpsA.
Keywords:
Non-ribosomal peptide synthetase
Indigoidine
BpsA
Natural products
Domain-specific inhibitors
2009 Elsevier Ltd. All rights reserved.
Modular synthases make up the core biosynthetic machinery
that produce essential small molecule metabolites such as fatty
acids, polyketides, and non-ribosomal peptides. Besides their
involvement in fatty acid anabolism throughout all kingdoms of
life, they are responsible for the production of bioactive
compounds crucial for cell survival in bacteria, fungi, and plants.
These molecules often possess antibiotic, immunosuppressive,
cytostatic, and cytotoxic activities and have gained considerable
attention in modern medicine and agriculture.1-3
blue pigment synthetase A (BpsA), the bacterial source of the
blue pigment indigoidine (Fig. 1).
Biosynthesis by modular synthases resembles an assembly-
line strategy unique among metabolic pathways. Each synthase
consists of individual domains possessing distinctive functions,
such as the recognition of the substrate (amino acid, acyl-
coenzyme A (acyl-CoA), or acyl-acyl carrier protein (acyl-ACP)
species), propagation of the growing chain, tailoring and
condensation of intermediates, and finally, release of the
product.4, 5 For non-ribosomal peptide synthetases (NRPS), the
basic components include adenylation (A), peptidyl carrier
protein (PCP), condensation (C) and thioesterase (TE) domains.6,
Figure 1. Pipeline for the biochemical characterization of a modular
pigment-producing synthase. After heterologous expression of the
synthase gene and purification of the protein, the activity of each
individual domain is inhibited by specific small molecules and analyzed
in vitro and in vivo by pigment formation. Additionally, mutant proteins
of each individual domain are generated and analysed subsequently.
Phylogenetic analysis provides further information on the diversity and
versatility of the synthase.
7
Due to their size and complexity, the study of modular
synthases remains challenging, including mechanism, structure,
and sometimes even identification of the final product. With this
report, we aim to demonstrate the dissection of a modular NRPS
to probe the activity of each domain, as well as the overall
activity of the ensemble. Here, we provide a case study of the

transferase (PPTase).14 The PPTase transfers the 4’-
phosphopantetheine moiety of the cofactor CoA onto a conserved
serine residue of the PCP, enabling the PCP to tether the natural
product via a labile thioester linkage. BpsA produces the blue
pigment indigoidine and this enzymatic transformation was
utilized for the development of novel reporter systems,15, 16 blue-
white screens in Streptomyces species,17, 18 a glutamine sensor,19
a
potential semiconductor component,20 and other microbiology
applications.21,
Recent engineering efforts have led to
22
genetically modified roses with a blue color,23 as well as
bioproduction of indigoidine at titers approaching 100 g/L.24
Additionally, within an International Genetically Engineered
Machine (iGEM) synthetic biology project, NRPS domain
swapping led to production of novel indigoidine-containing
peptides.25 The iGEM project also swapped the PCP domain with
with various other PCPs, resulting in large variability in the
amount of product. However, Owen et al. had previously co-
expressed a PCP replacement of BpsA with various carrier
proteins showing no (or severely reduced) indigoidine
formation.26 In contrast to this split system, Beer et al. found that
many of the PCP replacement mutants do produce indigoidine.
Here we tested three BpsA mutants from the Walsh lab that
encode for the carrier protein domain of TdiA,27 and observed no
activity (Fig. S11).25 It appears that the linker regions between
domains (here between Ox-A-PCP and PCP-TE) are crucial for
productive catalysis and stable protein expression.28
Despite all the work on the utilization of BpsA as a tool, the
mechanism of indigoidine biosynthesis remains unknown. Some
have suggested that flavin-dependent oxidation of L-Gln occurs
on the carrier protein prior to cyclization and dimerization,29
whereas others have proposed a mechanism in which cyclization
occurs first and oxidation triggers dimerization.12 Here, we
systematically assess the different domains of BpsA using both
chemical and genetic tools, in order to set the stage for
experiments that will elucidate the mechanism of this NRPS.
Figure 2. A) Genomic organization of BpsA (gene cluster: AB250063,
protein: BAE93896) in Streptomyces lavendulae subsp. lavendulae including
domain-specific inhibitors. orfA is
a
putative ribose-phosphate
pyrophosphokinase, bpsA codes for indigoidine synthase, bpsB for
a
regulator, and bpsC for a putative S-adenosylmethionine synthase. bpsA =
3,849 bp = 1,282 aa. B) 1 L-Gln-sulfamoyladenosine inhibitor of A-domain,
2 thiazole inhibitor of flavin-dependent Ox-domain, 3 Gln-pantetheinamide, a
substrate for in situ transformation into a CoA analog and subsequent loading
onto a carrier protein using a PPTase, 4 and 5 are sulfonylfluoride-containing
inhibitors of proteases and thioesterases and 6 is 6-NOBP, a general PPTase
inhibitor. * denotes that a domain mutant is available.
We expressed and purified flavin-bound apo-BpsA and post-
translationally modified the protein using CoA and the
promiscuous PPTase Sfp.30 Holo-BpsA showed robust activity in
vitro,12 and E. coli BL21 cells co-expressing Sfp and BpsA
produced indigoidine when grown with liquid or solid media
Supplying the double transformant with L-Gln in the growth
media boosts the production of the insoluble blue pigment, and
Mn2+ supplementation is superior to Mg2+, the latter presumably
involved in PPTase activity (Fig. S3).31
Although the blue pigment resulting from indigoidine was
first observed in 1890,8 the indigoidine synthase was only
discovered in 2002 as IndC in the plant pathogen Dickeya
dadantii.9 IndC is conserved in many organisms, and is annotated
as IndC, BpsA, IgiD, or as putative synthase (Fig. S1, S2).10 IndC
is a single protein multi-domain NRPS module, that utilizes two
molecules of L-glutamine as building blocks to produce the
indigoidine product.9 In 2007, a homologous synthase was
identified and characterized in Streptomyces lavendulae, called
blue pigment synthase A (BpsA). The biological role of these
blue pigment synthases remains speculative, even so many years
after their discovery. They appear to be present in many different
organisms, ranging from bacteria to eukaryotes, and their genes
are often cryptic, such as in the bacterial insect pathogen
Photorhabdus luminescens (Fig. S1,S2).11
To confirm the bioinformatic prediction of domains, we set
out to design and synthesize individual inhibitors for each
domain within BpsA. The A-domain is an adenylate-forming
enzyme that loads L-Gln onto the PCP.12 BpsA contains an
interrupted A domain, in which the Ox domain separates a large
N-terminal from a smaller C-terminal portion of the A domain
(Fig. 2B).32 Adenylate-forming enzymes, including aminoacyl-
tRNA synthetases, are inhibited by sulfamoyl-containing analogs
33-40
(abbreviated as AMS).13,
AMS compounds are effective
inhibitors of adenylating enzymes because they closely mimic the
short-lived aminoacyl-intermediate formed when the enzyme
catalyzes the condensation of an amino acid and AMP (derived
from ATP). We synthesized and analyzed 5’-O-(N-(L-
glutaminyl)-sulfamoyl)adenosine 1 as an inhibitor of BpsA. Only
addition of millimolar amounts of this inhibitor resulted in a
substantial decrease in the formation of blue pigment in vitro,
with an IC50 of 3.8 mM (Fig. 3A). The high concentrations
required suggest a competitive, non-covalent mechanism of
inhibition, as previously observed in inhibition of the
biosynthesis of mycobactin with salicyl-amino-sulfonamide-
In vitro experiments showed that BpsA can only accept L-Gln
over all proteinogenic amino acids, and that the synthase oxidizes
and dimerizes the amino acid.12 BpsA is a single module NRPS,
containing an oxidation (Ox) domain integrated into the A
domain, a peptidyl carrier protein (PCP), and a terminal
thioesterase (TE) domain (Fig. 2). The Ox domain harbors a
flavin cofactor, as shown by UV absorbance measurements,13 and
purified protein has the characteristic yellow coloration of
flavoproteins. Like all modular synthases, BpsA requires post-
translational modification by
a
4’-phosphopantetheinyl

adenosine.40 However, why 1 does not inhibit BpsA as efficiently
found that micromolar amounts are sufficient to fully inhibit the
enzyme when applied to E. coli expressing Sfp and BpsA (Fig.
4A).
The third domain of BpsA is a PCP, which is not an enzyme
but a carrier protein, making traditional enzymatic inhibition
impossible. However, our lab has developed a methodology to
attach non-hydrolyzable analogs of thioester bound substrates to
carrier proteins through the preparation of pantetheine analogs
elaborated by CoA biosynthetic enzymes and the promiscuous
PPTase Sfp.43 Facile synthesis of L-Gln-pantetheinamide 3
allowed for the labeling of the BpsA PCP with a non-
hydrolyzable substrate mimic. This crypto-BpsA showed no
activity in vitro (Fig. S4), and in direct competition assay
between CoA and L-Gln- pantetheinamide -CoA, decreased
BpsA activity was clearly observed when as the ratio of CoA to
L-Gln- pantetheinamide decreased (Fig. 3E).
In order to tether substrates to NRPSs for catalysis, all PCPs
require post-translational modification by a PPTase. PPTases
have only recently been recognized and investigated as potential
45
anti-microbial targets.44,
In our work on the discovery of
inhibitors for the B. subtilis PPTase, Sfp, we found the general
PPTase inhibitor 6-NOBP 6 shows modest inhibition against a
variety of PPTases.44, 46 Based on these findings Owen et al.15
used 6 as PPTase inhibitor as demonstrated by indigoidine
production with BpsA. Indeed, 6 inhibited BpsA with an IC50 of
25 µM. (Fig. 3B and SI Experimental Methods).
The fourth domain of BpsA is the TE, which presumably
releases the product (or an intermediate) from the PCP. While the
exact mechanism is unknown, release of 5-amino-pyridinedione,
followed by spontaneous dimerization of two products, forming
Figure 3. Inhibition of individual BpsA domains. A) Inhibition of the
PPTase Sfp by 5’-O-[N-(L-phenylalanyl)sulfamoyl] adenosine 1. B)
Inhibition of A domain by 6-NOBP 6. C) Inhibition of TE domain by PMSF
5 and D) AEBSF 4. E) Activity of BpsA by varying the ratio of CoA to the
non-hydrolysable L-Gln- pantetheinamide analog 3 during preincubation of
BpsA with Sfp and CoaA/C/D.
either colorless leuco-indigoidine or blue indigoidine, is a
29
proposed mechanism.12,
Numerous TE inhibitors have been
developed over the past decades that rely on the nucleophilicity
48
of the active site serine or cysteine.47, To inhibit the TE of
BpsA,
we
used
the
general
protease
inhibitor
phenylmethylsulfonyl fluoride (PMSF) 5 and a derivative of the
reactive phenylsulfonyl fluoride moiety, 4-(2-aminoethyl)
benzenesulfonyl fluoride (AEBSF) 4. Blue pigment formation
was abrogated by micromolar concentrations of either inhibitor,
suggesting that although its function is speculative, the TE
domain is critical for catalysis (Fig. 3C, 3D). Interestingly, 5
showed ten-fold greater inhibition of blue pigment production
than 4, indicating that steric effects or polarity of the substrates
that enter the TE is important.
as other sulfonamide inhibitors is unclear. Salicylic acid-
sulfonamide (Sal-AMS) inhibits the A-domains MbtA, BasE,
EntE and VibE at 9 nM, 90 nM, 0.2 and 0.1 µM, respectively.35
Luciferase is inhibited by dehydroluciferyl-AMS at 0.3 µM,33
and acyl-CoA ligase MenE by O-succinylbenzoic acid-AMS at 6
µM.13 Thus, it remains a question why we observe poor
inhibition of BpsA by L-Gln-AMS. We hypothesize that due to
its polarity, it has low affinity for the A-domain of BpsA and
perhaps uptake is poor. Alternatively, interrupted A-domains
might bind this class of inhibitor differently compared to
classical A-domains.
A BpsA mutant lacking the TE domain exhibits no turnover
when compared to wild type (Fig. S5A). In an effort to discern
the importance of the TE domain, we incubated the BpsA mutant
lacking the TE domain with a previously described type II
thioesterase, TycF.49 TycF is known to remove substrates from
the phopshopantetheine arm of PCPs. However, the TE mutant
was unable to produce blue pigment with or without TycF (Fig.
S5B), suggesting that the TE domain of BpsA directly
participates in the formation of indigoidine, and does not simply
hydrolyze the tethered substrate off of the PCP.
The second domain of BpsA is a flavin-dependent oxidation
domain. The Ox domain is most likely responsible for oxidizing
either glutamine tethered to the PCP or a cyclized glutamine
intermediate. Takahashi et al. demonstrated by point-mutational
analysis of the Ox domain that binding of the cofactor flavin
mononucleotide FMN is essential for indigoidine production.12
Other flavin-dependent oxidation domains in NRPSs have been
studied by the Walsh lab, including EpoB, the oxidation domain
involved in epothilone biosynthesis.41 EpoB oxidizes the dihydro
heterocyclic thiazolinyl ring to a heteroaromatic oxidation state,
forming methylthiazolylcarboxy-S-EpoB. The Ox domain of
BpsA shares 29% identity with EpoB (AAF62884) (Table S1).
Although some NRPS Ox-domains have been characterized, few
inhibitors of these oxidases are known. Inspired by the work of
Walsh and co-workers, we recently synthesized 4H-
benzo[d][1,3]oxathiin-2-one (BOTO) 2 as Ox-domain inhibitor
to investigate flavin-dependent oxidase domains.42 Here, we

A BLAST search of the BpsA TE domain results in hits (24%
homology, Table S3) in atromentin synthase50 and TdiA,51 in
which TEs are involved in cyclization/dimerization and
dimerization of two activated indolepyruvic acid monomers,
respectively. Fascinatingly, the human FAS (FASN) thioesterase
also appears as a potential hit, with 21% homology, whereas the
entire synthase has low identity (Table S4). All four TEs have
the conserved His residue (2481FASN), and the level of
sequence similarity is further demonstrated in the TE-domain
phylogenetic tree (Fig. S6). Constructing phylogenetic trees
based on A, PCP, Ox and TE domains (Fig. S7-S9 and Table
S1-S4), reveals that many putative synthases have similar
domains, and that homologs of the individual BpsA domains can
be found in very different synthases. Combined, this
bioinformatic data shows that although organisms and synthases
are evolutionarily far removed from each other, individual
domains show convergent evolution.
coli strain co-expressing BpsA and Sfp, in which BpsA activity
can be visualized by blue pigmentation of bacterial colonies on
agar plates. We spotted the inhibitors on sterile filter paper discs
that were placed on top of freshly spread E. coli cells
overexpressing both BpsA and Sfp (Fig. 4). Whereas the A-
domain inhibitor 1 shows activity in vitro, this molecule does not
show any effect in bacteria. It is known that some acyl-
sulfonamides cannot efficiently enter cells,38 and combined with
the high measured IC50 and high polarity, 1 is a poor in cellulo
inhibitor. Ox-domain inhibitor 2 shows a marked effect in
cellulo. As concentration of 2 was decreased, we observe a shift
from growth inhibition (close to the center of the disk) to growth
without formation of pigment to growth with blue pigment
production (Fig. 4A). Pantetheinamide 3 has no effect on the
formation of blue pigment when supplemented to E. coli
overexpressing BpsA/Sfp. PPTase inhibitor 6 shows toxicity, but
no clear absence of blue pigmentation, perhaps because 6 is a an
unspecific PPTase inhibitor and E. coli harbors an essential
PPTase that is also inhibited by 6-NOBP.46 The thioesterase
inhibitors 4 and 5 show severe toxicity to E. coli at high
concentrations. At lower concentrations, both general inhibitors
still exhibited toxicity, but the presence of white colonies could
be detected (Fig. 4C, D). Although only two of the inhibitors
tested here show promising results in vivo, this approach can be a
useful addition to our toolbox for the interrogation of synthases.
Taken together, this data shows that using this small toolbox
of inhibitors, we can quickly deduce the enzymatic activities of
the domains of an NRPS in vitro, and these tools can be applied
to both known and unknown pathways. From these
straightforward experiments, we learned that the interrupted A-
domain32 is functional and active, the Ox-domain is flavin-
dependent, the PCP-domain can be loaded with various unnatural
cargo in vitro, and the TE-domain is essential for blue pigment
production and is inhibited by cysteine-targeting inhibitors. We,
and others, can now apply these tools towards characterization of
the hypothetical enzymatic and/or non-enzymatic route to the
final product indigoidine. We are confident that such a systematic
approach opens up many new paths to precisely dissect the
individual steps of multi-domain synthases.
We have designed a systematic chemical and genetic approach
for interrogating the individual activities of each domain of a
large natural product synthase. Although current technology
facilitates bioinformatic discovery of genes and proteins involved
in biosynthesis of secondary metabolites,52 there is a clear need to
verify predictions with experimental data. Selective inhibition of
synthase domains will become a key component in the toolbox of
the chemical biologist for the functional elucidation of the
mechanism of assembly-line synthases.
Figure 4. Inhibition of pigment production of E. coli strains expressing
BpsA and Sfp. A) 2 inhibits the Ox-domain and shows a halo of alive, but
white colonies (1 µl of 1 M, 100 mM and 10 mM); B) 6 inhibits PPTases
and 10 µl spots of 10 mM, 1 mM and 0.1 mM show toxicity, but no
apparent white halo; both C) 4 (10 µl of 1 mM and 0.1 mM ) and D) and 5
(10 µl of 10 mM, 1 mM and 0.1 mM) show toxicity, but also the presence
of several white colonies surrounding the lower concentrations of inhibitor.
To verify our in vitro results, we characterized a set of BpsA
mutants designed with selectively inactivated domains. We
expressed and purified a BpsA mutant with a K598E mutation in
the Ox-domain, preventing the binding of flavin, leading to a
colorless-protein; a BpsA mutant with an excised TE domain; a
stand-alone Ox-domain; and BpsA mutants with the TdiA27 PCP
swapped into the synthase. We verified that each of these mutants
are inactive. To assess whether two BpsA proteins can act in
trans to produce indigoidine, a matrix of inactive mutants of
BpsA was made (Table S5, Fig. S10).No significant activity was
observed for any of the mutant combinations, indicating that the
L-Gln substrates loaded onto the PCP of BpsA are incapable of
interacting with catalytic domains on a different monomer of
BpsA to produce indigoidine.
Acknowledgments
Funding was provided from NIH GM095970. We thank Prof.
D. F. Ackerley for the BpsA plasmid construct, Prof. C. T. Walsh
and C. J. Balibar for the BpsA mutant plasmid constructs, Dr. X.
Huang and Dr. A. Mrse for assistance with NMR data collection,
and Dr. Y. Su for MS services.
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Supplementary Material
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Dissecting modular synthases through
inhibition: a complementary chemical and
genetic approach
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Christopher R. Vickery, Ian P. McCulloch, Eva C. Sonnenschein, Joris Beld , Joseph Noel and Michael D.
Burkart