Mechanistic studies on the substrate-tolerant lanthipeptide synthetase ProcM

Article abstract of DOI:10.1021/ja504692v

Lanthipeptides are a class of post-translationally modified peptide natural products. They contain lanthionine (Lan) and methyllanthionine (MeLan) residues, which generate cross-links and endow the peptides with various biological activities. The mechanism of a highly substrate-tolerant lanthipeptide synthetase, ProcM, was investigated herein. We report a hybrid ligation strategy to prepare a series of substrate analogues designed to address a number of mechanistic questions regarding catalysis by ProcM. The method utilizes expressed protein ligation to generate a C-terminal thioester of the leader peptide of ProcA, the substrate of ProcM. This thioester was ligated with a cysteine derivative that resulted in an alkyne at the C-terminus of the leader peptide. This alkyne in turn was used to conjugate the leader peptides to a variety of synthetic peptides by copper-catalyzed azide-alkyne cycloaddition. Using deuterium-labeled Ser and Thr in the substrate analogues thus prepared, dehydration by ProcM was established to occur from C-to-N-terminus for two different substrates. Cyclization also occurred with a specific order, which depended on the sequence of the substrate peptides. Furthermore, using orthogonal cysteine side-chain protection in the two semisynthetic peptide substrates, we were able to rule out spontaneous non-enzymatic cyclization events to explain the very high substrate tolerance of ProcM. finally, the enzyme was capable of exchanging protons at the α-carbon of MeLan, suggesting that ring formation could be reversible. These findings are discussed in the context of the mechanism of the substrate-tolerant ProcM, which may aid future efforts in lanthipeptide engineering.

Full text of DOI:10.1021/ja504692v

Article
pubs.acs.org/JACS
Mechanistic Studies on the Substrate-Tolerant Lanthipeptide
Synthetase ProcM
Subha Mukherjee and Wilfred A. van der Donk*
Department of Chemistry and Howard Hughes Medical Institute, University of Illinois at UrbanaChampaign, 600 South Mathews
Avenue, Urbana, Illinois 61801, United States
S
* Supporting Information
ABSTRACT: Lanthipeptides are a class of post-translationally
modified peptide natural products. They contain lanthionine
(Lan) and methyllanthionine (MeLan) residues, which
generate cross-links and endow the peptides with various
biological activities. The mechanism of a highly substrate-
tolerant lanthipeptide synthetase, ProcM, was investigated
herein. We report a hybrid ligation strategy to prepare a series
of substrate analogues designed to address a number of
mechanistic questions regarding catalysis by ProcM. The
method utilizes expressed protein ligation to generate a C-
terminal thioester of the leader peptide of ProcA, the substrate
of ProcM. This thioester was ligated with a cysteine derivative that resulted in an alkyne at the C-terminus of the leader peptide.
This alkyne in turn was used to conjugate the leader peptides to a variety of synthetic peptides by copper-catalyzed azide−alkyne
cycloaddition. Using deuterium-labeled Ser and Thr in the substrate analogues thus prepared, dehydration by ProcM was
established to occur from C-to-N-terminus for two different substrates. Cyclization also occurred with a specific order, which
depended on the sequence of the substrate peptides. Furthermore, using orthogonal cysteine side-chain protection in the two
semisynthetic peptide substrates, we were able to rule out spontaneous non-enzymatic cyclization events to explain the very high
substrate tolerance of ProcM. Finally, the enzyme was capable of exchanging protons at the α-carbon of MeLan, suggesting that
ring formation could be reversible. These findings are discussed in the context of the mechanism of the substrate-tolerant ProcM,
which may aid future efforts in lanthipeptide engineering.
leader peptide to produce the final lanthipeptide (Figure 1A).1
Lan and MeLan formation involves first the dehydration of Ser
and Thr residues to generate dehydroalanine (Dha) and
INTRODUCTION
■
Lanthipeptides are a class of ribosomally synthesized and post-
translationally modified peptides (RiPPs1) that contain the
dehydrobutyrine (Dhb), respectively. The dehydrated residues
characteristic thioether residues lanthionine (Lan) and methyl-
lanthionine (MeLan).2,3 A subclass of lanthipeptides with
then undergo Michael-type addition by the side-chain thiol of
antibacterial activity is known as lantibiotics,4 which are
cysteines to generate the thioethers Lan and MeLan,
respectively (Figure 1B).22
effective against many Gram-positive bacteria including some
drug-resistant species.5 For instance, nisin has been used for
Prochlorosins are a large group of lanthipeptides produced
by marine cyanobacteria of the genus Prochlorococcus. In
Prochlorococcus MIT9313, a single enzyme, ProcM, catalyzes
the post-translational modification of 29 different substrates
over 50 years as a preservative in the food industry to combat
food-borne pathogens without significant bacterial resistance.6
Lanthipeptides that do not display antibacterial activity can
exert antiviral,7 antiallodynic,8 antinociceptive,9 and morpho-
(ProcAs), thereby generating many distinct thioether ring
genic10 activities. Cyclic peptides such as lanthipeptides are also
topologies within the 29 prochlorosin products (Pcns;
Supporting Information Figure S1).23 As such, the Pcn
increasingly recognized as promising compounds for disrupting
protein−protein interactions.11−16 Investigation of the synthe-
tases that post-translationally generate lanthipeptides would aid
biosynthetic system is intriguing with respect to the details of
thioether ring formation that may explain the remarkable
diversity of the products formed by ProcM. A previous study
suggested that the enzyme might generate a subset of the
in engineering efforts to produce molecules with desirable
properties.17−19
thioether rings, which would then preorganize the substrate to
During lanthipeptide biosynthesis, the ribosomal machinery
facilitate non-enzymatic cyclization of the other rings,23 as
illustrated schematically for a substrate with two thioether rings
first synthesizes linear precursor peptides called LanAs.3 The
LanA peptide comprises an N-terminal leader peptide that is
believed to serve several possible roles, including recognition by
the synthetase,20,21 and a C-terminal core peptide that is post-
translationally modified. Proteolytic cleavage then removes the
Received: May 10, 2014
© XXXX American Chemical Society
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thioether rings, which ultimately results in the ring topology
with the lowest free energy. On the other hand, kinetic control
would lead to thioether ring formation involving the lowest
activation energy barrier. A previous study on the biomimetic
cyclization of nisin’s B-ring suggested that non-enzymatic
thioether ring formation is governed by kinetic control and that
the Michael-type addition is irreversible.29 In the current study,
the reversibility of cyclization in the presence of ProcM was
investigated.
RESULTS
■
Choice of Substrates. Two core peptides were chosen for
investigation out of the repertoire of 29 possible substrates. The
first choice was ProcA2.8, which is transformed into pro-
chlorosin (Pcn) 2.8, a product with two non-overlapping
lanthionine rings (Figure 2A). In the majority of the
Figure 1. (A) Generic scheme for lanthipeptide biosynthesis. (B)
Common post-translational modifications in lanthipeptides.
in Figure S2. Investigation of the stereochemistry of the
thioether rings in a subset of Pcns showed that they all had the
DL stereochemistry (2S,6R-Lan and 2S,3S,6R-MeLan),24 iden-
tical to what has been observed for the majority of
lanthipeptides analyzed to date.22 Though this conserved
stereochemistry of Michael-type addition suggested enzymatic
cyclization, non-enzymatic cyclization of preorganized inter-
mediates with high stereoselectivity could not be ruled out
because in previous biomimetic studies of lanthipeptide
biosynthesis non-enzymatic cyclization also took place with
high selectivity for the DL stereoisomers.25−27 In this study, we
experimentally probed a potential role of non-enzymatic
cyclization in prochlorosin maturation.
The substrate-tolerant synthetase ProcM dehydrates core
peptides containing a variety of sequences with different
residues flanking Ser and Thr and also with different numbers
of intervening residues (Figure S1). Currently, it is not known
whether ProcM dehydrates its substrates in a directional
fashion and whether any observed directionality is general for
different substrates. In this study, we expanded upon a
previously reported strategy28 to assign the directionality of
dehydration of substrates by ProcM. Additional experiments
also revealed the order of ProcM-catalyzed cyclization of these
substrates.
Figure 2. Structures of prochlorosins 2.8 and 3.3. The fragments of the
Lan/MeLan residues originating from Cys are in red, and those
originating from Ser/Thr residues are in blue.
lanthipeptides discovered to date, the thioether rings are
installed by nucleophilic attack of cysteines onto dehydrated
residues that are located toward the N-terminus. However, in
prochlorosins, the thioether rings are formed by Cys residues
located on either side of the dehydrated residues, as illustrated
for Pcn2.8 generated from ProcA2.8 (Figure 2; see also Figure
S1). Thus, studies with Pcn2.8 could reveal whether perhaps
enzymatic cyclization forms rings in one direction and non-
enzymatic cyclization forms thioether rings in the opposite
direction. In addition, we selected Pcn3.3, a compound
containing overlapping thioether rings, to probe the effect of
substrate preorganization on non-enzymatic ring formation
(Figure 2B). As described below, for both substrates, we
determined the order of dehydration and cyclization and
investigated the possibility of non-enzymatic cyclization and
reversibility as determinants of the ring topology.
ProcM Dehydrates ProcA2.8 Precursor Peptide in C-
to-N-Terminal Fashion. Previous studies on LanM lanthi-
peptide synthetases showed that dehydration is directional,
moving from the N-terminus to the C-terminus of the
substrate.30 To investigate whether this would also be the
case for the substrate-tolerant ProcM, we used the method
In the 29 prochlorosins with diverse ring topologies
produced by ProcM, often a single ring structure out of several
possible topologies is observed (Figure S1B).23,24 The observed
high site selectivity in prochlorosin cyclization could be the
result of either thermodynamic or kinetic control. Thermo-
dynamic control would require the reversible installation of
developed by Sussmuth and co-workers,28 in which specific Ser
̈
residues are replaced by 2,3,3-deuterium-labeled serine. In such
substrates, dehydration of unlabeled Ser involves loss of 18 Da
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(−H2O) while dehydration of the labeled serine results in loss
of 19 Da (−HDO; Figure 3A,B). The lanthipeptide synthetase
3 spanning residues 3−19 were synthesized by SPPS with Ser13
or Ser9 replaced with 2,3,3-deuterium-labeled Ser (Figure 4).
Native chemical ligation of 2 and 3 with ProcA2.8 leader-AA-
thioester 1 afforded the substrates 4 and 5 (Figure 5A,C).
These substrates were treated with ProcM, and after various
time points, ProcM was removed from a portion of the assay by
ultrafiltration. The filtrate was incubated with endoproteinase
GluC to remove most of the leader peptide, and the digest was
analyzed by matrix-assisted laser desorption/ionization time-of-
flight (MALDI-TOF) and electrospray ionization (ESI) mass
spectrometry. With both substrates, the ESI and MALDI-TOF
mass spectrometric analyses showed that Ser13 was dehydrated
prior to Ser9 (Figure 5B,D and Figures S3 and S4).
Figure 3. Strategy for determination of directionality of dehydration
using [2,3,3-2H]-Ser or [2,3-2H]-Thr residues. (A) Dehydration of Ser
incurs a loss of 18 Da. (B) Dehydration of labeled Ser results in a loss
of 19 Da. (C) Dehydration of Thr incurs a loss of 18 Da. (D)
Dehydration of labeled Thr results in a loss of 19 Da. By tracking
which dehydration involves a loss of 19 Da, directionality of
dehydration can be established.28
Although EPL worked well to determine the directionality of
dehydration of ProcA2.8, other substrates do not always have a
conveniently located Cys. Hence, we evaluated another hybrid
ligation strategy based on both EPL and copper-catalyzed
alkyne−azide cycloaddition (CuAAC).32,33 In previous studies
on lacticin 481 synthetase, an alkyne-containing leader peptide
was generated by chemical synthesis,34,35 but in this work, we
elected to generate the much longer ProcA leader peptide by
heterologous expression in Escherichia coli as a fusion protein to
an intein and chitin-binding domain.36 Given the high-sequence
similarity of the leader peptides of prochlorosin precursors
(Figure S1), the ProcA3.2 leader peptide was arbitrarily chosen
for the designed precursor peptides. The C-terminal Gly of the
ProcA3.2 leader peptide was mutated to Lys to allow efficient
cleavage of the peptide thioester linkage to the chitin resin by 2-
mercaptoethanesulfonate sodium salt (MESNa),37 and to
introduce a LysC endoproteinase cleavage site C-terminal to
the leader peptide. EPL of the MES-thioester with (R)2-amino-
N-(but-3-yn-1-yl)-3-mercaptopropionamide (6, Figure 6) gen-
erated the leader peptide with a C-terminal alkyne modification
(Figure S5). The cysteine residue incorporated during EPL was
protected with iodoacetamide to prevent any potential
interference in the enzymatic cyclization reaction (Figure S5).
The core peptides of ProcA2.8 were then synthesized by
SPPS, again incorporating deuterium-labeled Ser at positions 9
and 13. In the last step prior to cleaving the peptides from the
resin, the CuAAC ligation handles 7 or 8 (Figure 6) were
coupled to their N-termini. The building block 7 would enable
convenient removal of the leader peptide by photolysis,38
whereas building block 8 was expected to improve the
efficiency of the ligation owing to the copper ligating ability
of the pyridine ligand.39 Two ProcA2.8 precursor peptides 9
and 10 were thus synthesized by CuAAC to probe
directionality of dehydration (Figure 7A,C). The substrates
were treated with ProcM, and at several time points, ProcM
was removed from a portion of the assay by ultrafiltration; the
filtrate was incubated with protease LysC and the digest
analyzed by MALDI-TOF MS (Figures S6 and S7). With both
substrates 9 and 10, the analysis showed that Ser13 was
dehydrated prior to Ser9, indicating C-to-N-terminal dehy-
dration (Figures 7B,D). This observation demonstrated that the
presence of a triazole linker in the designed substrate did not
affect the directionality of dehydration. Furthermore, GC−MS
analysis of the product revealed the presence of Lan rings of the
same DL stereochemistry as in the wild-type (wt) product
(Figure S8), thus suggesting that ProcM correctly recognizes
the substrate with a triazole linker between leader and core
peptides. The observation that the substrate analogue generated
by CuAAC furnished the same product and with the same
directionality as with native substrate enabled us to extend our
ProcM requires an N-terminal leader peptide for maturation of
the precursor peptide.23 The highly conserved leader peptide is
ca. 65 amino acid residues long, and the highly variable core
peptides comprise between 13 and 32 amino acid residues
(Figure S1), resulting in a precursor peptide that is too long to
prepare conveniently by linear solid phase peptide synthesis
(SPPS). Instead, we used expressed protein ligation (EPL)31 to
generate full-length precursor peptides containing deuterium-
labeled serine. The ProcA2.8 core peptide has a cysteine at
position 3 that can be used for EPL. Peptide 1 corresponding to
the ProcA2.8 leader peptide with two additional Ala residues
from the N-terminus of the ProcA2.8 core peptide was
generated with a peptide thioester at the C-terminus using
intein chemistry (Figure 4). Two ProcA2.8 core peptides 2 and
Figure 4. Schematic representation of ProcA2.8 leader-AA-MESNa
thioester (1), ProcA2.8 core peptide Δ1−2 with Ser13 replaced with
[2,3,3-2H]-Ser (2), and ProcA2.8 core peptide Δ1−2 with Ser9
replaced with [2,3,3-2H]-Ser (3).
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Figure 5. Directionality of dehydration of wt ProcA2.8. (A) Structure of ProcA2.8 precursor peptide 4 assembled using EPL with Ser13 replaced
with [2,3,3-2H]-Ser. (B) MALDI-TOF MS of 4 partially dehydrated by ProcM and digested by GluC. (C) Structure of ProcA2.8 precursor peptide 5
assembled using EPL with Ser9 replaced with [2,3,3-2H]-Ser. (D) MALDI-TOF MS of 5 partially dehydrated by ProcM and digested by GluC.
(Figure 8B,D and Figures S9 and S10), in agreement with the
overall C-to-N-terminal dehydration observed with ProcA2.8.
To confirm that the observed directionality of dehydration of
wt ProcA3.3 was the same as that of the analogues 12 and 13,
tandem MS was used to analyze partially dehydrated wild-type
ProcA3.3. The order of dehydration of Thr11 and Thr18
cannot be distinguished by this method because of the
overlapping rings of prochlorosin 3.3, but the timing of
dehydration of Thr3 can be determined. Using ultra-
performance liquid chromatography (UPLC) coupled with
ESI-MS, peptide ions with predominantly two-fold dehydration
were analyzed (Figure S11A). The ion was fragmented, and the
observed fragment ions demonstrated that Thr3 escaped
dehydration in the two-fold dehydrated ProcA3.3 (Figure
S11B), in agreement with the conclusion that we reached from
studies on the triazole-containing ProcA3.3 precursor peptide
(i.e., Thr3 is dehydrated last).
Directionality of Cyclization by ProcM. ProcM was
incubated with expressed and purified His6-ProcA2.8 obtained
as reported previously,23 and the assay was quenched by
removing ProcM at various time points. After incubation with
endoproteinase LysC to remove most of the leader peptide, the
digest was treated with iodoacetamide (IAA). Any noncyclized
thiol would react with IAA, and the enzymatic assay conditions
were optimized to allow buildup of an incompletely cyclized
intermediate that resulted in one IAA adduct (Figure S12A).
Figure 6. Structure of building block 6 used to attach an alkyne at the
C-terminus of the leader peptide by EPL. Building block 7 was used
for installation of a photocleavable triazole linker between the leader
and the core peptides. The building block 8 was used to facilitate the
click reaction via a copper-chelating group.
investigation of directionality of dehydration to other substrates
where the sequence of the core peptide does not allow EPL to
assemble the native precursor peptide.
Directionality of Dehydration of ProcA3.3 by ProcM.
To probe directionality of dehydration in prochlorosins
containing threonine residues and overlapping rings, we
prepared the ProcA3.3 derivatives 12 and 13 with either
Thr18 or Thr11 replaced with [2,3-2H]-Thr, respectively
(Figure 8A,C). The peptides were treated with ProcM and
after various time points, the enzyme was removed by
ultrafiltration. The filtrate was incubated with endoproteinase
GluC to remove most of the leader peptide, and the digest was
analyzed by MALDI-TOF MS. The data demonstrated that
Thr18 was dehydrated first followed by Thr11 and Thr3
Figure 7. Directionality of dehydration in ProcA2.8 analogues. (A) Structure of ProcA2.8 precursor peptide 9 with Ser13 replaced with [2,3,3-2H]-
Ser. (B) MALDI-TOF MS of 9 partially dehydrated by ProcM and after LysC cleavage. (C) Structure of ProcA2.8 precursor peptide 10 with Ser9
replaced with [2,3,3-2H]-Ser. (D) MALDI-TOF MS of 10 partially dehydrated by ProcM and after LysC cleavage.
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Figure 8. Directionality of dehydration of ProcA3.3 analogues containing a triazole linker. (A) Structure of ProcA3.3 analogue 12 with Thr18
replaced with [2,3-2H]-Thr. (B) MALDI-TOF MS of 12 partially dehydrated by ProcM and digested by GluC. (C) Structure of ProcA3.3 analogue
13 with Thr11 replaced with [2,3-2H]-Thr. (D) MALDI-TOF MS of 13 partially dehydrated by ProcM and digested by GluC.
Figure 9. Probing non-enzymatic cyclization in ProcA2.8. (A) Non-enzymatic cyclization of the A-ring in the presence of the enzymatically
preinstalled B-ring. Reagents and conditions: (i) HEPES, ATP, TCEP, MgCl2, substrate 14 (50 μM), ProcM (30 μM); (ii) ProcM was removed, the
intermediate 18 was desalted and lyophilized, dissolved in 0.1% TFA, and irradiated; (iii) the peptide was dissolved in HEPES buffer (pH 8.0) to
allow non-enzymatic cyclization followed by treatment with iodoacetamide. (B) MALDI-TOF MS analysis showed that non-enzymatic cyclization
was slow and incomplete, as indicated by the presence of IAA adduct 22. (C) Non-enzymatic cyclization of the B-ring in ProcA2.8 in the presence of
enzymatically preinstalled A-ring. Reagents and conditions: (i) HEPES, ATP, TCEP, MgCl2, ProcM (30 μM), substrate 15 (50 μM); (ii) ProcM was
removed, intermediate 19 was irradiated with UV light and lyophilized; (iii) the lyophilized peptide was dissolved in solution containing all
components in (i) except ProcM, digested with LysC and treated with excess iodoacetamide. (D) MALDI-TOF MS analysis showed that non-
enzymatic cyclization was incomplete, as indicated by the presence of IAA adduct 23.
ESI-MS/MS was performed on this peptide, and Cys3 was
found to be alkylated (Figure S12B). The fragmentation
pattern suggested that the B-ring had formed in this
intermediate. Hence, cyclization of Cys19 occurred prior to
Cys3, suggesting C-to-N-terminal directionality of cyclization.
ProcM was also incubated with His6-ProcA3.3, and the assay
was quenched by removing ProcM at various time points. After
endoproteinase LysC digestion, the peptides were treated with
IAA. The ProcM assay conditions were again optimized to trap
an intermediate that resulted in one IAA adduct (Figure S13A).
The observed fragmentation pattern of this peptide by ESI-
MS/MS showed that Cys21 was alkylated, suggesting that
Cys14 had formed a thioether ring with Dhb18 (Figure S13B).
The data suggest that cyclization of ProcA3.3 may not take
place with C-to-N-terminal directionality, but that instead the
MeLan ring between Cys14 and Dhb18 is formed in the
monocyclic intermediate (see Figure 2 for the Pcn3.3 ring
topology).
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Figure 10. Probing non-enzymatic cyclization in ProcA3.3. (A) Non-enzymatic cyclization of the A-ring in the presence of enzymatically preinstalled
B-ring. Reagents and conditions: (i) HEPES, ATP, TCEP, MgCl2, substrate 16 (50 μM), ProcM (20 μM); (ii) ProcM was removed and the
intermediate 20 was irradiated with UV light and lyophilized; (iii) the lyophilized peptide was dissolved in assay solution containing all components
in (i) with or without ProcM, digested by LysC, and treated with iodoacetamide. (B) MALDI-TOF MS of solution obtained after treatment in (iii).
(C) ProcM assay of substrate 17 generated a mixture of intermediates 21 and 25. (D) ProcM was removed from the mixture of 21 and 25, and the
product mixture was irradiated with UV light and lyophilized. The peptide mixture was dissolved in solution containing all ProcM assay components
with or without ProcM. The products were then treated with LysC and IAA. The MALDI-TOF MS spectra of the two assays are presented.
Substrate Design To Probe Non-enzymatic Cycliza-
tion in Prochlorosin Maturation. To address whether non-
enzymatic cyclization assists ProcM in the maturation of
substrates with variable ring topologies, substrates were
designed that restrict ProcM installation to selected thioether
rings by orthogonal protection of a subset of cysteine thiols in
the core peptide. Release of the protecting group would
generate an intermediate that would allow investigation of non-
enzymatic cyclization in the presence of other preinstalled
thioether rings.
A variety of orthogonal protecting groups for the cysteine
thiols were tested. tert-Butyl disulfide protection proved not
suitable because removal by reducing agents like tris(2-
carboxyethyl)phosphine (TCEP) and tributylphosphine
formed adducts with dehydrated residues. Conversely,
acetamidomethyl protection was unsuccessful because its
removal required oxidizing agents like iodine, which partially
oxidized the thioether rings. However, the o-nitrobenzyl and
4,5-dimethoxy-o-nitrobenzyl groups were readily introduced
into the peptide during SPPS40 and were cleanly released by
UV irradiation (365 nm).
Hence, four precursor peptides were prepared using the
previously described hybrid EPL/CuAAC ligation strategy (14
and 15 in Figure 9 and 16 and 17 in Figure 10), which were
treated with ProcM to produce the intermediates 18−21 and
25. Photorelease of ortho-nitrobenzyl protecting groups then
generated substrates to probe non-enzymatic cyclizations of
each of the rings in ProcA2.8 and ProcA3.3 in the presence of
other enzymatically installed rings.
Efficient Cyclization of ProcA2.8 Requires ProcM.
ProcM correctly processed the ProcA2.8 precursor peptide
analogue 14 to generate the B-ring and a dehydroalanine at the
ninth position of the core peptide (intermediate 18) (Figure
9A) as demonstrated by tandem MS (Figure S14). The peptide
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18 was irradiated at 365 nm to release the o-nitrobenzyl group
on Cys3 along with the leader peptide that was attached via
linker 7. The resulting peptide was incubated in buffered
solution at pH 8.0 to probe non-enzymatic cyclization under
conditions where enzymatic cyclization is complete. The
peptide was then treated with IAA to report on noncyclized
Cys residues. MALDI-TOF MS analysis revealed the
predominant presence of IAA adduct 22, which indicated
incomplete non-enzymatic cyclization of the A-ring of pro-
chlorosin 2.8 (Figure 9B). Thus, non-enzymatic cyclization of
the A-ring in the presence of the B-ring is much slower than
enzymatic cyclization. Non-enzymatic cyclization was also
conducted at higher pH (pH 8.5) and for a longer time period
(12 h), but non-enzymatic cyclization of the A-ring still did not
proceed to completion.
ProcM converted the ProcA2.8 analogue 15 (Figure 9C) to
an intermediate 19 that contained the A-ring of prochlorosin
2.8 and a dehydroalanine at the 13th position as evidenced by
tandem MS (Figure S15). The peptide 19 was irradiated to
remove the o-nitrobenzyl group from Cys19 and was incubated
with ProcM. The peptide was then treated with LysC to
remove the leader peptide and with IAA to probe cyclization.
Analysis by MALDI-TOF MS showed that, whereas the
enzymatic cyclization under these conditions is complete in 1
h, the non-enzymatic cyclization still resulted in a mixture of
cyclized product and IAA adduct 23 after 16 h (Figure 9D).
Because the observed non-enzymatic cyclization of the B-ring
was very slow compared to enzymatic cyclization, these
experiments do not provide support for efficient non-enzymatic
ring formation facilitated by preorganization upon formation of
one of the rings.
Ring Formation in ProcA3.3 Requires ProcM. Treat-
ment of the ProcA3.3 precursor analogue 16 with ProcM
resulted in the formation of a product containing a dehydro-
butyrine (Dhb) at positions 3 and 11 and a MeLan formed
between Cys14 and Dhb18 (Figure 10A), as confirmed by
tandem MS after LysC cleavage of 20 (Figure S16). The o-
nitrobenzyl group was removed from Cys21 by UV irradiation,
and the peptide was subjected to both enzymatic and non-
enzymatic assay conditions. Following the cyclization assays,
the peptides were digested with endoproteinase LysC and
treated with IAA. The enzymatic assay did not show IAA
adduct formation, whereas the non-enzymatic assay showed
predominant formation of the IAA adduct 24 (Figure 10A) as
demonstrated by MALDI-TOF MS (Figure 10B). Hence, non-
enzymatic cyclization of the A-ring of Pcn3.3 in a peptide that
already contained the B-ring is much slower than when this
process is catalyzed by ProcM. Thus, the experiments with
peptides 18−20 show that non-enzymatic cyclization is too
slow to be kinetically competent to be a part of the overall
process.
The product obtained after ProcM treatment of the ProcA3.3
precursor analogue 17 was not a single peptide but a mixture of
peptides 21 and 25 with two different ring topologies (Figure
10C). The major product 21 had a MeLan formed between
Cys21 and Dhb18 and the minor product 25 contained a
MeLan formed between Cys21 and Dhb11 (as seen in native
modified ProcA3.3, Figure 2), as evidenced by tandem ESI-MS
on the individual products that were separated on analytical
scale by UPLC (Figure S17A,B). These findings can be
explained on the basis of the order of cyclization of ProcA3.3
discussed above. In wt ProcA3.3, Cys14 forms a ring with
Dhb18 in the observed monocyclic intermediate, and hence
only Dhb11 is available to Cys21 for cyclization. However, in
peptide 17, Cys14 is protected, and therefore, both Dhb11 and
Dhb18 are available for reaction with Cys21; apparently the
enzyme then prefers formation of the smaller ring between
Cys21 and Dhb18. Compounds 21 and 25 could not be
separated on preparative scale, and therefore, the mixture was
subjected to UV irradiation to release the o-nitrobenzyl group
from Cys14. The mixture of deprotected peptides was
subsequently subjected to both enzymatic and non-enzymatic
cyclization assay conditions. The peptides were then digested
with endoproteinase LysC followed by treatment with IAA.
The non-enzymatic assay resulted in the formation of IAA
adduct, thus indicating the absence of significant spontaneous
non-enzymatic cyclization in the mixture of two peptides,
whereas the enzymatic assay resulted in complete cyclization as
evidenced by the lack of IAA adduct (Figure 10D). The
cyclized product mixture was separated on analytical scale by
UPLC and tandem MS analysis revealed the identity of the two
products obtained from 21 and 25. In addition to the
overlapping ring topology as observed in Pcn3.3 (Figure
S18A), an alternate non-overlapping ring topology was formed
(Figure S18B). Hence, manipulation of the order of cyclization
by using Cys protecting groups allows access to a ring topology
not seen with the native substrate.
Reversibility of Thioether Ring Formation. To test
whether thioether ring installation in prochlorosins is reversible,
ProcA substrates were modified by ProcM in D2O, resulting in
incorporation of one deuterium at each newly formed α-
stereocenter of Lan/MeLan. The modified precursor peptides
were then purified and subjected to standard ProcM assay
conditions in unlabeled aqueous buffer. If the Michael-type
addition is reversible, then the incorporated deuterium would
be expected to exchange with a protium in the assay in
unlabeled buffer (Figure 11).
Figure 11. Scheme for the exchange assay. Substrate is modified by
ProcM in deuterium-labeled buffer to generate product with one
deuterium per Lan/MeLan. Exchange of the installed deuterium was
then investigated by treating the modified precursor peptide with
ProcM in unlabeled buffer. Exchange can occur by abstraction of
deuterium followed by protonation (solid blue arrows) or by retro-
Michael-type addition (dashed blue arrow) and recyclization.
His-tagged ProcA2.8 was heterologously expressed in E. coli
and purified and subjected to ProcM with all assay components
dissolved in D2O. ProcM was removed by ultrafiltration, and
the peptide was desalted and lyophilized. A portion of the
lyophilized peptide was digested with endoproteinase GluC to
generate the modified core peptide with a five amino acid
overhang at its N-terminus originating from the leader peptide
(Figure 12A). As anticipated, both ESI and MALDI-TOF MS
analysis demonstrated the incorporation of two deuterium
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digested with GluC before mass spectrometric analysis. The
modified ProcA3.3 treated in aqueous buffer without ProcM
did not result in any exchange (Figure 12D, blue), but the
modified substrate treated in aqueous buffer with ProcM
showed exchange of both deuterium atoms with protium
(Figure 12D, red). Analysis of the exchange over time at two
different concentrations of ProcM revealed that the exchange
was time-dependent and dependent on the concentration of
ProcM (Figure S19). Also, exchange of one deuterium was
significantly faster, with the second deuterium exchange
requiring higher enzyme concentration and longer incubation
time. Under the conditions of the assay (100 μM modified
ProcA3.3 and 5 μM ProcM), the exchange of the first
deuterium was already detected after short incubation times
(15 min; Figure S19). The dehydration of ProcA3.3 catalyzed
by ProcM under the same conditions is complete at this time
point, but cyclization is still incomplete. Hence, the observed
deuterium exchange appears kinetically competent with the
cyclization process.
D−H Exchange Is Not Observed in ProcA2.8 Sub-
strates with Ser Mutated to Thr. Given the observation of
D−H exchange in modified ProcA3.3 but not ProcA2.8, it is
interesting to note the differences between the two substrates.
Pcn3.3 contains overlapping rings whereas Pcn2.8 does not,
and the rings in Pcn3.3 are formed by MeLan residues whereas
both rings in Pcn2.8 are formed by Lan residues. To investigate
if the lack of exchange in cyclized ProcA2.8 was because it
contains Lan and not MeLan, two mutants of ProcA2.8 were
generated by site-directed mutagenesisone with Ser9
replaced with Thr (ProcA2.8-S9T) and another with Ser13
replaced with Thr (ProcA2.8-S13T). Both substrates were
modified by ProcM in D2O, incorporating two deuterium
atoms in the process. The purified products were subjected to
the D−H exchange assay conditions and were digested with
GluC. Subsequent analysis by MALDI-TOF MS revealed that
D−H exchange had not occurred (Figure S20A−D). Hence, in
ProcA2.8 with two non-overlapping rings, even changing a Lan
to a MeLan does not lead to D−H exchange, suggesting that
ring topology may instead dictate whether ProcM can exchange
the α-proton of the cross-linked amino acids.
Exchange in ProcA3.3 Involves the B-Ring. As noted
above, the exchange process for cyclized ProcA3.3 involves one
deuterium that was exchanged relatively rapidly with a proton
from solvent, whereas the second deuterium exchange was
much slower. Because cyclization of ProcA3.3 results in a ring
within a ring, tandem MS could not be used to determine
which of the two MeLan residues is associated with the faster
exchange. We therefore mutated Thr11 to Ser such that
cyclization would result in one Lan and one MeLan, which in
principle can be distinguished by GC−MS after acidic
hydrolysis of the product. Thus, ProcA3.3-T11S was first
incubated with ProcM under the standard conditions in D2O.
The resulting product was purified and shown to contain two
deuterium atoms by mass spectrometry (Figure S21B). The
labeled peptide was then treated with ProcM in unlabeled
buffer, resulting in relatively rapid exchange of one deuterium
(Figure S21C), similar to the observations with wild-type
ProcA3.3. The resulting peptide containing one deuterium was
then hydrolyzed, and the Lan and MeLan residues were
derivatized as previously described.41,42 Analysis by GC−MS
resulted in detection of unlabeled derivatized MeLan and
deuterium-labeled derivatized Lan (Figure S22). Hence, the
relatively fast exchange occurs in the MeLan in ring B. Both
Figure 12. Deuterium incorporation and exchange in ProcA2.8 and
ProcA3.3. (A) Structure of GluC cleaved ProcA2.8 core peptide
fragment incorporating two deuteriums. Residues in purple originate
from the leader peptide. (B) MALDI-TOF MS of GluC cleaved
fragment of deuterium-labeled ProcA2.8 (50 μM) after assay without
(blue trace) and with ProcM (20 μM) treatment in aqueous buffer
(red trace). (C) GluC digested ProcA3.3 core peptide incorporating
two deuteriums. Residues in purple originate from the leader peptide.
(D) MALDI-TOF MS of GluC digested fragment of modified
deuterium-labeled ProcA3.3 (100 μM) without aqueous ProcM
treatment (blue trace) and with aqueous ProcM (40 μM) treatment
(red trace).
atoms. The full-length peptide was then incubated in unlabeled
buffer in the presence or absence of ProcM for 17 h. Exchange
of deuterium with protium was not observed (Figure 12B, red).
Hence D−H exchange did not occur in ProcA2.8 containing
two non-overlapping lanthionine rings, suggesting that such
rings are not installed reversibly for this peptide.
ProcM-Cyclized ProcA3.3 Undergoes Enzymatic D−H
Exchange. To also explore a substrate containing overlapping
rings, ProcA3.3 was heterologously expressed and purified. The
peptide was modified by ProcM in D2O, the enzyme was
removed from the modified peptide by ultrafiltration, and the
product peptide was desalted and lyophilized. Analysis by
MALDI-TOF MS after endoproteinase GluC digestion
demonstrated incorporation of two deuterium atoms as
expected (Figure 12C). The full-length peptide was then
incubated in unlabeled buffer with or without ProcM and
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Lan and MeLan residues had the correct DL stereochemistry as
confirmed by co-injection with synthetic standards (Figure
S23).
suggest that enzymatic deprotonation at the α-position of
MeLan residues does occur in some rings. Furthermore,
although true kinetic studies on the enzyme have thus far been
unsuccessful because of the many reactions it catalyzes and the
absence of a convenient and quantitative method to measure
each step, the general time dependence of exchange and
cyclization of ProcA3.3 suggests that deprotonation at the α-
carbon of MeLan might be kinetically competent. It is
important to note that the D−H exchange assay reports on
the reversibility of the protonation of the enolate during MeLan
formation; it does not necessarily indicate that the cyclization is
reversible (i.e., a reversible Michael-type reaction). Attempts to
trap a free thiol formed from ring opening involving Lan/
MeLan residues were not successful.
The observation that the deuterium in the smaller B-ring
exchanges faster than the larger A-ring is somewhat puzzling. If
deuterium exchange indeed reports on reversibility of ring
formation, then one might have expected that the ring that
forms last (A-ring) would open up first. Several explanations
may account for the observation that D−H exchange instead
occurs in the B-ring. One possibility is that the formation of the
two rings are entirely independent, that is, that the kinetics of
forming either ring is independent of whether the other ring is
already formed (Figure S24). In this scenario, an intermediate
with the smaller B-ring installed would indeed be observed in
the forward process, and exchange of the same ring could be
favored in the reverse process (Figure S24). Although it appears
unlikely that ring formation would be entirely independent in a
short peptide with overlapping rings such as the ProcA3.3 core
peptide, we cannot rule out this possibility. A second possibility
is that the intermediate with the B-ring that is observed during
the ring formation process is not a productive intermediate en
route to the final product. It is possible that formation of the
final product requires the A-ring to be formed first (Figure
S25). In that scenario, the intermediate with the B-ring formed
would be in equilibrium with the starting peptide, and an
intermediate with the A-ring installed would not be detected
because of very rapid formation of the final product from this
intermediate (Figure S25). This model would also readily
explain why it is the B-ring that undergoes exchange in the
reverse direction. We cannot unambiguously distinguish
between both hypotheses, but they do make predictions that
can be tested in future studies.
DISCUSSION
■
In an effort to understand the remarkable substrate tolerance of
ProcM, four aspects of the lanthionine synthetase were
investigated. Aided by a hybrid ligation protocol that allowed
us to install synthetic core peptides onto a heterologously
expressed leader peptide, we investigated the directionality of
both dehydration and cyclization, the possibility that a non-
enzymatic cyclization step might account for the high diversity
of ring topologies of the products, and the possibility of
reversibility of ring formation. Use of labeled ProcA substrate
unequivocally demonstrated that ProcM dehydrates two very
different substrate peptides in a C-to-N-terminal fashion. This
same directionality was also reported for the class III
lanthipeptide synthetase LabKC43 and another RiPP synthetase
BalhCD,44 whereas for other class II lanthipeptides, such as
LctM and HalM2, the directionality of dehydration was
reported to be N-to-C-terminal based on tandem MS.30 The
latter directionality is more readily explained using a proximity
effect if the active site for dehydration is close to the leader
peptide binding site. Such a model might be expected to lead to
an erosion of strict directionality if Ser/Thr residues are close in
sequence space, which indeed has been observed.30 Explanation
of a C-to-N-terminal directionality of dehydration as observed
here and in LabKC and BalhCD requires a more complex
model. Although we cannot completely rule out that the
directionality of dehydration by ProcM is simply reflecting the
reactivity of each individual site as a result of different flanking
residues or secondary structure, the lack of any sequence
similarity in the two peptides that are both shown to be
dehydrated in C-to-N-terminal direction in this study leads us
to favor an explanation that involves a specific juxtaposition of
the leader and core peptide binding sites that favors
dehydration of C-terminal residues. Structural studies will be
required to provide further information.
Interestingly, the cyclization of the two peptides also
occurred with a specific order, but this order was not
necessarily directional. Whereas for ProcA2.8 the Cys that is
located closer to the C-terminus reacted first, in ProcA3.3, it
was the Cys that was closer to the N-terminus that appeared to
react first. In the latter substrate, this results in the smaller B-
ring being formed in the observed intermediate. Preference for
formation of a smaller ring was also observed when Cys14 of
ProcA3.3 was protected, resulting in Cys21 forming a ring that
is not seen in the normal product. Hence, it appears that the
order of cyclization catalyzed by ProcM is determined more by
the ring size than by directionality of the enzyme. This
observation agrees with other recent studies that suggest that
the precursor peptides may have an inherent bias for formation
of certain rings.45,46
The remarkable substrate tolerance of ProcM suggested the
possibility that perhaps only a subset of the rings are generated
enzymatically and that these enzymatically formed macrocycles
preorganize the peptide for subsequent non-enzymatic
cyclization. However, our current data show that, for two
different substrates, non-enzymatic cyclization of intermediates
that contain one ring is too slow to be kinetically competent for
the enzymatic process. Another question that has not been
previously addressed in lanthipeptide biosynthesis is whether
thioether ring formation is reversible or not. Our experiments
CONCLUSIONS
■
This study shows that dehydration of ProcA2.8 and ProcA3.3
by ProcM takes place with C-to-N directionality, and that for
these two substrates, cyclization is also an ordered process but
that the order is determined by the ring topology. Furthermore,
non-enzymatic cyclization is shown not to be involved in the
formation of two very different ring topologies in prochlorosins
2.8 and 3.3. Whether these observations are general for all
ProcA substrates remains to be determined. Finally, for some
ring topologies, the protonation of the enolate during the
Michael-type addition is reversible (i.e., the enzyme can remove
the α-proton from the cyclized product), which possibly may
indicate that the enzyme can also catalyze a retro-Michael
addition. Future studies will focus on investigating this
hypothesis.
I
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Journal of the American Chemical Society
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ASSOCIATED CONTENT
* Supporting Information
Synthetic procedures and characterization of compounds and
peptides, biochemical assay and molecular biology procedures,
and supporting figures. This material is available free of charge
via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
vddonk@illinois.edu
Notes
■
(18) Molloy, E. M.; Ross, R. P.; Hill, C. Biochem. Soc. Trans. 2012, 40,
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(19) Field, D.; Hill, C.; Cotter, P. D.; Ross, R. P. Mol. Microbiol.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors thank Dr. Yanxiang Shi for providing the pET15b
plasmids encoding genes for ProcA2.8, ProcA3.3, and ProcA3.2,
Dr. Christopher Thibodeaux for providing the pTXB1 plasmid
encoding the ProcA2.8 leader-AlaAla fused to intein-CBD,
Manuel Ortega for assistance with protein purification, and Dr.
Alexander V. Ulanov for help during GC−MS analysis. This
study was supported by the National Institutes of Health (GM
058822) to W.A.V. A Bruker UltrafleXtreme MALDI TOF/
TOF mass spectrometer was purchased in part with a grant
from the National Institutes of Health (S10 RR027109).
(27) Zhou, H.; van der Donk, W. A. Org. Lett. 2002, 4, 1335.
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