Thiopeptide Pyridine Synthase TbtD Catalyzes an Intermolecular Formal Aza-Diels-Alder Reaction

Article abstract of DOI:10.1021/jacs.8b11852

Thiopeptide pyridine synthases catalyze a multistep reaction involving a unique and nonspontaneous intramolecular aza-[4 + 2] cycloaddition between two dehydroalanines to forge a trisubstituted pyridine core. We discovered that the in vitro activity of pyridine synthases from the thiocillin and thiomuracin pathways are significantly enhanced by general base catalysis and that this broadly expands the enzymes substrate tolerance. Remarkably, TbtD is competent to perform an intermolecular cyclization in addition to its cognate intramolecular reaction, underscoring its versatility as a biocatalyst. These data provide evidence that pyridine synthases use a two-site substrate recognition model to engage and process their substrates.

Full text of DOI:10.1021/jacs.8b11852

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Communication
Thiopeptide Pyridine Synthase TbtD Catalyzes an
Intermolecular Formal Aza-Diels-Alder Reaction
Jonathan W. Bogart, and Albert A. Bowers
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 17 Jan 2019
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Thiopeptide Pyridine Synthase TbtD Catalyzes an
Intermolecular Formal Aza-Diels-Alder Reaction
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Jonathan W. Bogart and Albert A. Bowers*
Division of Chemical Biology and Medicinal Chemistry Eshelman School of Pharmacy, and Lineberger Comprehensive
Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Supporting Information Placeholder
ABSTRACT: Thiopeptide pyridine synthases catalyze a
multistep reaction involving a unique and non-spontaneous
intramolecular aza-[4+2] cycloaddition between two
dehydroalanines to forge a tri-substituted pyridine core. We
discovered that the in vitro activity of pyridine synthases
from the thiocillin and thiomuracin pathways are
significantly enhanced by general base catalysis and that
this broadly expands the enzymes substrate tolerance.
Remarkably, TbtD is competent to perform an
intermolecular cyclization in addition to its cognate
intramolecular reaction, underscoring its versatility as a
biocatalyst. These data provide evidence that pyridine
synthases use a two-site substrate recognition model to
engage and process their substrates.
The Diels-Alder (DA) reaction has been a staple of
organic synthesis since its discovery by Otto Diels and Kurt
Alder in 1928.1 Numerous total syntheses have exploited
different versions of this typically concerted cycloaddition
reaction and related transformations to efficiently construct
carbon frameworks and stitch together complex organic
scaffolds.2 In recent years, it has become apparent that
similar chemistry is employed widely in the biosynthetic
pathways of complex natural products.3–6 Several enzymes
have been found to catalyze DA or DA-like reactions,
including PyrI4 in pyroindomycin biosynthesis, SpnF in
spinosyn biosynthesis, and LovB in lovastatin biosynthesis,
among others.7–11 In many of these cases, the enzymes
work to activate, accelerate, and/or direct the
stereochemical outcome of an otherwise spontaneous
cycloaddition reaction. As a result, reactions catalyzed by
Figure 1. (a) Proposed mechanism of pyridine synthases.
these Diels-Alderases can be highly substrate dependent
Steps potentially influenced by base (1-3) are highlighted.
and their biocatalytic utility is limited.
(b) Thiopeptides thiocillin (left) and thiomuracin (right)
Pyridine synthases in thiopeptide biosynthesis provide
and their precursor peptide sequences (c, d). Orange re-
unique examples of what is thought to be an enzyme
gions highlight pyridine synthase recognition sequences
catalyzed aza-DA. The biosyntheses of thiopeptides are
used in this work. Core residues in red are converted to
similar to other ribosomally synthesized and post-
Dhas/Dhbs and residues in blue are converted to thiazoles.
translationally modified peptides (RiPPs) in that the C-
stage formation of the class-defining nitrogenous
heterocycles at the core of the thiopeptide
pharmacophores.14-16 The key reaction is thought to entail
an intramolecular formal [4+2] cycloaddition between two
terminal ‘core’ of a precursor peptide is transformed into
the mature natural product by a suite of peptide modifying
enzymes that are recruited by an N-terminal ‘leader’
peptide.12,13 Pyridine synthases are responsible for the late
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dehydroalanines (Dhas) followed by elimination of water
measured whereas values of 3.9 and 7.1μM were observed
at pH 8.0 and 9.0 respectively. These data suggest that the
pH does not significantly influence substrate binding.
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and leader peptide (LP) to yield a tri-substituted pyridine
core (Fig. 1a).17,18 In contrast to other DAs, this reaction is
non-spontaneous and incurs a significant thermodynamic
penalty due to the inactivated nature of the reaction
subcomponents. Synthetic versions of this cycloaddition
are low yielding and require high temperatures.19
The in vivo manipulation of thiopeptide biosynthetic
gene clusters has demonstrated that pyridine synthases are
very promiscuous enzymes and can be used to generate a
variety of thiopeptide derivatives with different sized
macrocycles and pyridine substituents.20–25 More
specifically, enzymatic macrocyclization is independent of
any structural preorganization imposed by the core peptide,
as neither its length nor rigidity prevent cyclization.23 Thus
we reasoned that pyridine synthases might be able to
catalyze an intermolecular reaction between isolated 2π
and 4π components. Such a reaction would provide perhaps
the most extreme example of substrate promiscuity and
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support
a two-site model of substrate recognition.
Therefore, we sought to test whether pyridine synthases
TclM or TbtD, from the biosynthetic pathways of thiocillin
(1) and thiomuracin (2), respectively, could catalyze an
intermolecular reaction (Fig. 1b). Optimization of the
enzyme reaction conditions was an important initial step to
test this hypothesis because our previous work showed that
the enzymes could be slow and several modifications
known to be permitted in vivo either refused to be cyclized
in vitro and/or generated by-products.26
Figure 2. Simplified TclM (a) and TbtD substrates (b). (c)
The rate of TclM or TbtD catalyzed pyridine formation is
enhanced under basic pH. (b) Impact of pH on kcat and Kd.
We first looked to improve the in vitro activity of TclM
and TbtD by investigating the influence of pH on the rate
of pyridine formation. Several steps of the proposed
mechanism could be influenced by pH: (1) tautomerization
of the enamide to iminol, (2) elimination of water and (3)
aromatization (Fig. 1a). We used our previously described
solid-phase strategy to prepare substrates for TclM and
TbtD (compounds 3 and 4, respectively, Fig. 2a, b) based
on their respective precursor peptides, TclE and TbtA (Fig.
1c).26 Each substrate consisted of a simplified core with a
minimal LP fragment that was previously found to be
sufficient for complete processing.14,26 The TclM substrate
(3), was synthesized with a 10-residue LP, while the TbtD
substrate (4), had a 15-residue LP (Fig. 2a, b). Enzymatic
reactions were monitored at neutral and basic pH by
measuring the distinctive absorbance of the newly forming,
tri-substituted pyridine at 350nm over time. Reaction rates
were calculated based on standard curve (see SI). A
significant increase in rate was observed for each enzyme
at pH 9.0 (Fig. 2c). Specifically for TbtD, a nearly 10-fold
increase in kcat was observed from pH 7.2 to 9.0 (Fig. 2d,
S1). Although kcat at pH 9.0 is still generally considered
low, it is comparable to other late stage RIPP-modifying
enzymes.27–32
Due to the pronounced binding affinity of TbtA’s N-
terminal 16-residue LP for TbtD,33 we sought to determine
whether this region of the LP might further enhance the
reaction rate by allosteric regulation in trans. Studies have
shown that this region contribute much of the full-length
leader LP binding affinity to TbtD (Kd = 1.3μM),33 but is
not strictly essential for enzymatic processing.16,26 The 16-
residue N-terminal fragment (6) was synthesized by SPPS
and reaction rate for substrate 4 was measured at pH 7.2
and 9.0 in presence or absence of 6. No appreciable change
in rate was observed (Fig. S3). These data suggest that the
N-terminal LP fragment does not act as an allosteric
modulator. In the context of full length TbtA, this
fragment’s higher affinity may allow the LP to bind first,
thereby increasing local concentration of the core around
the active site to facilitate faster modification.
As an initial test of TbtD’s improved substrate
promiscuity under the optimized reaction conditions we
assessed its ability to cyclize TbtA LP mutants. Previously,
alanine scanning mutagenesis had been used to investigate
LP binding to TbtD16,33 but not substrate processing. We
reasoned that similar experiments might identify residues
within the LP that are important for catalysis in addition to
acting as a metric for promiscuity. A suite of 16, largely
alanine-substituted, LP variants were prepared in parallel
by solid-phase peptide synthesis (SPPS, see SI). The LP
variants were separately tested at pH 7.2 and 9.0, and
LCMS analysis revealed several differences (Table 1, Fig.
S4a-q). Overall, 12 of the 16 substrates were processed at
neutral pH, while 15 were cyclized under basic pH.
Next, we assessed whether pH influenced substrate
binding. We measured the dissociation constant, Kd for
TbtA bearing the 15-residue LP and MBP-TbtD via
fluorescence polarization assay. A non-reactive substrate
for TbtD, with alanines instead of Dhas, was synthesized
and derivatized with fluorescein isothiocyanate (FITC) at
the N-terminus (5). Kd’s were determined at pH 7.2, 8.0
and 9.0 (Fig. 2d, S2). Effects of pH on Kd were minor
compared to those on kcat. At pH 7.2, a Kd of 3.2μM was
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Interestingly, only the H27A LP variant failed to cyclize
under either condition. These data indicate that TbtD
exhibits broader substrate promiscuity under basic pH.
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Table 1. Cyclization efficiency of TbtA LP mutants.
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With the enhanced activity of pyridine synthases
confirmed, we sought to test whether TclM or TbtD could
catalyze an intermolecular reaction. 2π components for
both TclM and TbtD were prepared by SPPS and consisted
of the corresponding minimal leader sequences with a C-
terminal Dha-thiazole. The TclM 4π component was
synthesized via SPPS following the same synthetic strategy
that was used to prepare the intramolecular substrates. The
TbtD 4ꢀ component was synthesized in solution from
thiazole building blocks following standard peptide
coupling procedures (see SI). In either case, each 4ꢀ
component was designed to present the amide bond and
Dha proposed to be involved in the cycloaddition plus two-
to-three modified residues flanking either side. Incubation
of each set of substrates with their respective pyridine
synthases at pH 7.2 for 21 hrs yielded only trace amounts
of product by LCMS. At pH 9.0, we again observed only
trace amounts of product in reactions with TclM (Table
S1). However, TbtD cleanly consumes both components
Figure 3. (a) TbtD-catalyzed intermolecular reaction. (b-d)
UV traces of 2π and 4π substrates after 20 h in the pres-
ence (c and d) and absence (b) of TbtD.
calculated based on LCMS analysis and the rate of
cyclization was measured by change in absorbance at 350
nm over time relative to a standard curve of purified
product (Fig. 4b, S5a-h, S6). The results illustrate several
key
requirements
for
intermolecular
substrates
(summarized in Fig. 4a): (1) the C-terminal heterocycle
must be present in the 2π component (Fig. 4b, entry 3), but
a thiazole can be exchanged for an oxazole without
significantly perturbing the rate or conversion (Fig. 4b,
entry 2); (2) TbtD is sensitive to changes to both the N- and
C-termini of the 4π component (comparing entry 1 to 6, 7
and 8 in Fig. 4b); (3) the bisazole motif is critical for
cyclization; truncation to a single thiazole results in no
product (Fig. 4b, entry 8); (4) removal of the C-terminal
Dha results in no product – this Dha can be replaced with
an alanine, albeit at significant cost to overall rate (Fig. 4b,
entries 6 and 7); (5) in all 4π substrates, exchanging
thiazoles for oxazoles is tolerated but there is a decrease in
efficiency in each case (comparing entry 1 to 2, 4 and 5 in
Fig. 4b). Ultimately, all permissible reactions could be
forced to full conversion with higher enzyme loadings,
allowing isolation and structural confirmation of products
and produces
a new UV peak with characteristic
absorbance at 350 nm and [m/z] consistent with a fully
formed pyridine product (Fig. 3, S5a). The intermolecular
reaction was scaled up and the identity of the product was
confirmed by 1H and 13C NMR. TbtD is capable of
recognizing the 2π and 4π reaction partners separately,
without need for tethering between them, suggesting that an
exceptionally broad array of macrocycles could likely be
prepared by this enzyme.
In order to further define the limits of the TbtD-catalyzed
intermolecular reaction, we prepared a small library of 2π
and 4π substrates. With respect to the 2π component,
previous LP truncation experiments and the above LP
variants have defined the important residues in the LP so
we focused further variation on the C-terminal side on the
Dha. The thiazole (Thz) was changed to an oxazole (Oxz,
Fig. 4b, entry 2) or removed entirely (Fig. 4b, entry 3).
These 4π substrates were again synthesized via solution-
phase chemistry and Dhas were installed either through
alkylation-elimination chemistry of cysteine thiols or via
mesylation-elimination of serine alcohols (see SI). As with
the 2π substrates, thiazoles were iteratively changed to
oxazoles and residues flanking the necessary Dha were
removed to isolate the crucial components of the 4π
substrate. For each new reaction, a percent conversion was
1
by H and 13C NMR. These data demonstrate TbtD is
tolerant of major changes to nearly every residue outside of
the two Dhas undergoing pyridine ring formation.
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Funding Sources
1
2
No competing financial interests have been declared.
3
4
ACKNOWLEDGMENT
We thank N. J. Kramer, R. Wolfenden, C. Neumann and B.
Li for informative discussions. JWB is recipient of an
AFPE pre-doctoral fellowship and a GSK pre-doctoral fel-
lowship. This work was supported by NIH Grant
GM125005 (AAB). AAB is a Beckman Young Investigator
and acknowledges support by Arnold & Mabel Beckman
Foundation.
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Figure 4. Substrate scope of the intermolecular reaction.
(a) Consistent with intramolecular substrate 4, a 15-residue
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thiazoles, O for oxazoles). Removing residues with red
triangles prevents cyclization.
In conclusion, we found that the thiomuracin pyridine
synthase TbtD is capable of catalyzing an intermolecular
pyridine formation in addition to its cognate intramolecular
reaction. The intermolecular reaction proved possible only
under rate-enhancing alkaline conditions, which allow an
approximate 10-fold increase in enzyme turnover (kcat)
while minimally affecting substrate binding (Kd). The
thiocillin pyridine synthase, TclM, observed similar base-
catalyzed rate-enhancement but could not perform an
intermolecular reaction, suggesting that different families
of pyridine synthases may have distinct substrate
requirements. The intermolecular chemistry of TbtD
provides evidence that pyridine synthases use a two-site
model to engage and process their precursor peptides; this
reaction is a strong indicator of this enzyme family’s broad
promiscuity, suggesting that their use as biocatalysts could
be extended beyond natural thiopeptide scaffolds, to allow
larger ring sizes, incorporation of non-natural functionality
or even novel linear substrates. The intermolecular
reaction may be of use in elucidating the mechanism of
these enzymes and may provide a barometer for evaluating
new pyridine synthases for biotechnology applications.
ASSOCIATED CONTENT
Experimental details, synthetic schemes, figures available
at http:// pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
abower2@email.unc.edu
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