Mechanistic Investigations of PoyD, a Radical S-Adenosyl- l -methionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis

Article abstract of DOI:10.1021/jacs.7b08402

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of bioactive peptides. Among RiPPs, the bacterial toxin polytheonamide A is characterized by a unique set of post-translational modifications catalyzed by novel radical S-adenosyl-l-methionine (SAM) enzymes. Here we show that the radical SAM enzyme PoyD catalyzes in vitro polytheonamide epimerization in a C-to-N directional manner. By combining mutagenesis experiments with labeling studies and investigating the enzyme substrate promiscuity, we deciphered in detail the mechanism of PoyD. We notably identified a critical cysteine residue as a likely key H atom donor and demonstrated that PoyD belongs to a distinct family of radical SAM peptidyl epimerases. In addition, our study shows that the core peptide directly influences the epimerization pattern allowing for production of peptides with unnatural epimerization patterns.

Full text of DOI:10.1021/jacs.7b08402

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Article
Mechanistic investigations of PoyD, a radical SAM enzyme catalyzing
iterative and directional epimerizations in polytheonamide A biosynthesis
Aubérie Parent, Alhosna Benjdia, Alain Guillot, Xavier Kubiak, Clémence
Balty, Benjamin Lefranc, Jérôme Leprince, and Olivier Berteau
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08402 • Publication Date (Web): 18 Dec 2017
Downloaded from http://pubs.acs.org on December 18, 2017
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Journal of the American Chemical Society
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Mechanistic investigations of PoyD, a radical SAM enzyme catalyz-
ing iterative and directional epimerizations in polytheonamide A bio-
synthesis
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Aubérie Parent1+, Alhosna Benjdia1+, Alain Guillot1+, Xavier Kubiak1+, Clémence Balty1, Benjamin
Lefranc2, Jérôme Leprince2 & Olivier Berteau1*
1 Micalis Institute, ChemSyBio, INRA, AgroParisTech, Université ParisꢀSaclay, 78350 JouyꢀenꢀJosas, France
2 Inserm U1239, PRIMACEN, University of Rouen Normandy, Fꢀ76000 Rouen, France
KEYWORDS. radical S-adenosyl-L-methionine, radical SAM, radical AdoMet, iron-sulfur cluster, polytheonamide, RiPP
ABSTRACT: Ribosomallyꢀsynthesized and postꢀtranslationallyꢀmodified peptides (RiPPs) are a growing family of bioactive pepꢀ
tides. Among RiPPs, the bacterial toxin polytheonamide A is characterized by a unique set of postꢀtranslational modifications cataꢀ
lyzed by novel radical SAM enzymes. Here we show that the radical SAM enzyme PoyD catalyzes in vitro polytheonamide epimerꢀ
ization in a CꢀtoꢀN directional manner. By combining mutagenesis experiments with labeling studies and investigating the enꢀ
zyme’s substrate promiscuity, we deciphered in details the mechanism of PoyD. We notably identified a critical cysteine residue as
a likely key Hꢀatom donor and demonstrated that PoyD belongs to a distinct family of radical SAM peptidyl epimerases. In addiꢀ
tion, our study shows that the core peptide directly influences the epimerization pattern allowing for production of peptides with
unnatural epimerization patterns.
INTRODUCTION
Among RiPPs, polytheonamide A is so far unique by
requiring three radical SAM enzymes (PoyB, PoyC and PoyD)
to introduce two types of postꢀtranslational modifications (i.e.
methylation and epimerization)17. Another fascinating feature
of polytheonamide A is the extent of postꢀtranslational modifiꢀ
cations introduced by these three enzymes. Indeed, the two
B12ꢀdependent radical SAM enzymes: PoyC and PoyB, have
been recently shown in vitro15 and in vivo23 to be responsible
for the formation of the thirteen Cβ methylation and the Nꢀ
terminal terꢀbutyl group (Figure 1a). By coꢀexpressing PoyD
with various truncated forms of the precursor peptide PoyA in
E. coli17, 23, it has been shown that PoyD catalyzes the eighteen
epimerizations found in polytheonamide A in a likely CꢀtoꢀN
directionality (Figure 1). Based on its sequence and these
unique properties, PoyD has been predicted to form a distinct
class of radical SAM enzymes2, 5, 24. To understand the mechaꢀ
nism of this enzyme and unravel how it introduces a unique
pattern of epimerizations within a peptide backbone, we unꢀ
dertook the biochemical characterization of the radical SAM
enzyme PoyD.
Ribosomallyꢀsynthesized and postꢀtranslationallyꢀmodified
peptides (RiPPs) form an expanding family of natural products
which was recently unified1ꢀ2. This large family of natural
products contains diverse classes of peptides including lanꢀ
thipeptides, thiopeptides and microcins which have relevant
biological properties including notably antibiotic and antiꢀ
cancer activities. These fascinating biological properties are
one of the reasons behind the renewed interest in RiPPs. Inꢀ
deed, RiPPs appear as promising natural products to address
the antibiotic resistance crisis but also as a source of novel
molecules to regulate the human microbiota3ꢀ5.
RiPPs are produced according to a simple biosynꢀ
thetic logic, a precursor peptide containing a leader or a folꢀ
lower sequence is synthesized and modified to various extent
by tailoring enzymes, before being generally secreted and the
leader (or follower) cleaved off6ꢀ7. RiPPs have been shown to
contain a wealth of postꢀtranslational modifications such as
thioether8ꢀ9 and carbonꢀcarbon10ꢀ12 bonds, unusual Cꢀ
methylation13ꢀ16 and epimerization5, 17. In a unique manner, the
soꢀcalled radical SAM enzymes, an emerging superfamily of
metalloꢀenzymes4, 18ꢀ19 have been shown to catalyze all these
various and chemically unrelated modifications2. Indeed,
radical SAM enzymes, despite a core mechanism involving
RESULTS
PoyD is a radical SAM enzyme catalyzing in vitro pep-
tide epimerization. PoyD was expressed as a Strepꢀtag fusion
protein in E. coli (Figure 2a). The purified protein exhibited
the typical brownish color of ironꢀsulfur enzymes and, after
anaerobic ironꢀsulfur reconstitution, UVꢀvisible analysis
showed an increase in the absorption bands at 320 nm and 420
the coordination of SꢀadenosylꢀLꢀmethionine (SAM) to an
[4Feꢀ4S]2+/1+ cluster in a bidendate fashion20ꢀ21 and the generaꢀ
tion of the 5'ꢀdeoxyadenosyl radical (5'ꢀdA•)22 to initiate catalꢀ
ysis, have evolved an unsurpassed but still illꢀunderstood
diversity of mechanisms and reactions.
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nm, consistent with an increase of the ironꢀsulfur cluster conꢀ resis and mass spectrometry. As shown, we were able to exꢀ
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tent of the protein (Figure 2b). Determination of the iron
content indicated that asꢀpurified PoyD contained 1.1± 0.1 mol
of Fe per polypeptide. After anaerobic reconstitution, PoyD
contained 4.1± 0.4 mol of Fe per polypeptide. These results
supported that PoyD contained one [4Feꢀ4S] cluster per monꢀ
omer.
press and purify PoyA even in the absence of PoyD, with
purity similar to previous reports17, 23 (Supplementary Figure
S1). LCꢀMS/MS analysis of the amino acid content of PoyA,
after acid hydrolysis and derivatization with Nꢀαꢀ(2,4ꢀdinitroꢀ
5ꢀfluorophenyl)ꢀLꢀvalinamide (
Methods) showed, as expected, that PoyA contained
acid residues when coꢀexpressed with PoyD. We notably
identified ꢀAsn (converted as ꢀAsp during the hydrolysis
process) and ꢀVal, which are characteristic of polytheonaꢀ
mide A17 (Figures 2c). PoyA, expressed in the absence of
PoyD, did not contained ꢀamino acid residues. Unfortunateꢀ
LꢀFDVA) (see Supplementary
Dꢀamino
D
D
D
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D
ly, unmodified PoyA exhibited very poor solubility in aqueous
buffers and proved to be an impracticable substrate for the in
vitro study of PoyD.
We have recently shown that a peptide derived from the
core sequence of PoyA (residues 1 to 49, Figures 1a & b) and
containing the residues +1 to +15, could serve as substrates for
PoyC, the B12ꢀdependent radical SAM enzyme catalyzing
valine Cꢀmethylation15. However, because of the high content
of hydrophobic residues (i.e. Ile, Val and Ala), we had to
insert an Nꢀterminus stretch of Lys residues to obtain a soluble
substrate. With this substrate, PoyC catalyzed methylation of
Valꢀ14 but not of the five other Val residues located between
positions +5 to +10, presumably because of the presence of the
Lysꢀstretch.
Interestingly, the leader sequence of PoyA contains charged
amino acid residues (Asp) that could be exploited to improve
the solubility of the peptides and make them more suitable for
LCꢀMS analysis. We thus synthesized PoyA derivatives conꢀ
taining residues ꢀ9 to ꢀ1 from the leader sequence and residues
+1 to +10 from the core region (Figure 1c). To simplify the
detection and analyses by HPLC and LCꢀMS, we also introꢀ
duced either one Nꢀterminal Trp residue (Peptide 1) or substiꢀ
tuted the residue Gln ꢀ5 by a Lys moiety (Peptide 2) (Figure
1c). Each peptide was assayed with PoyD under anaerobic and
Figure 1 – Structure of Polytheonamide A and peptide sub-
strates designed to investigate PoyD mechanism. (a) Structure
of polytheonamide A. Numbers indicates amino acid residues
location. Methyl groups labeled in blue are inserted by the radial
SAM enzyme PoyC, while methyl groups labeled in purple have
been proposed to be inserted by the radical SAM enzyme PoyB.
reducing conditions in the presence of the SꢀadenosylꢀLꢀ
methionine (SAM) cofactor.
Red labels are Dꢀamino acid residues formed by the radical SAM
enzyme PoyD. (b) Sequence of PoyA, the peptide precursor of
polytheonamide A. Circles filled in blue indicate the amino acid
residues epimerized in mature polytheonamide A. The enzymes
responsible for postꢀtranslational modifications of PoyA are indiꢀ
cated next to the arrows. (c) Sequence of peptides 1 & 2 used as
substrates. Circles filled in grey indicate amino acid residues
from the leader sequence while circles filled in white indicate
amino acids from the core sequence. White circles with a red line
are residues epimerized in polytheonamide A. Circles filled in red
indicate amino acid residues introduce in the sequence for analytiꢀ
cal purpose. Numbers are relative to PoyA sequence with positive
numbers for the core peptide and negative numbers for the leaderꢀ
peptide sequence.
To assess the activity of PoyD, we tried to produce PoyA,
the polytheonamide A precursor and proposed substrate of
PoyD (Figure 1b), in E. coli. Previous studies have pointed
out that PoyA cannot be expressed in the absence of PoyD,
suggesting a role of foldase/chaperone for this latter17, 23. We
thus expressed PoyA as a Hisꢀtag fusion protein in the presꢀ
ence of PoyD. However, in order to obtain an unmodified
PoyA, we also attempted to express PoyA in the absence of
PoyD. PoyA was then purified under denaturating conditions
(See Supplementary Methods) and analyzed by gel electrophoꢀ
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These results indicated that two deuterium atoms were inꢀ
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troduced into peptides 4 & 5 while three deuterium atoms
were introduced into peptide 6 during the reaction. LCꢀ
MS/MS analysis of peptide 4 showed that one deuterium atom
was located in Alaꢀ8 and another one in Valꢀ10 (Figure 3b,
Supplementary Figure S3 & Tables S1 & S2). Analysis of
peptide 5 revealed a different pattern with one deuterium
atom introduced into Valꢀ9 and one into Valꢀ7 (Supplemen-
tary Figure S4a & Table S3). Finally, peptide 6 proved to
contain three deuterium atoms located in Valꢀ5, Valꢀ7 and
Valꢀ9 (Supplementary Figure S4b & Table S4). The modiꢀ
fied peptides were further purified by HPLC and their amino
acid content analyzed by LCꢀMS/MS (see Supplementary
Methods). Comparison with authentic standards showed that,
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in each peptide produced (i.e. peptides 4, 5 & 6),
DꢀVal and
DꢀAla residues were present (Figure 3c & Supplementary
Figure S5). These results established that peptides 4, 5 and 6
are diastereoisomers of peptide 1 and that PoyD is a peptidyl
epimerase which requires only an [4Feꢀ4S] cluster and the
SAM cofactor to convert in vitro LꢀVal and LꢀAla into their Dꢀ
configured counterparts.
PoyD substrate specificity. As shown above, incubation of
peptide 1 led to the formation of three peptides with two
epimerization patterns (i.e. natural pattern: Alaꢀ8 and Valꢀ10
and unnatural pattern: Valꢀ5, Valꢀ7 and Valꢀ9) involving eiꢀ
ther the Cꢀterminal or the penultimate residue with alternating
epimerizations, one of two residues (Figures 3a & b). With
peptide 2, only one new peptide was produced, albeit at low
level (Supplementary Figure S6). This novel peptide (pep-
tide 3) had a mass increment of +3 Da when the reaction was
performed in deuterated buffer, consistent with the incorporaꢀ
tion of three deuterium atoms. LCꢀMS/MS analysis allowed to
position deuterium incorporation into Valꢀ6, Alaꢀ8 and Valꢀ10
(i.e. the natural epimerization pattern) (Figure 3b, Supple-
mentary Figure S7 & Tables S5 & S6).
Figure 2 – In vitro characterization of PoyD. (a) Gel electro-
phoresis analysis of purified PoyD expressed in E. coli. MW:
Molecular weight markers. (b) UV-visible spectrum of as-
purified (dotted line) and anaerobically reconstituted PoyD
(plain line). (c) HPLC analysis of the amino acid content of
PoyA after its in vivo expression in E. coli alone or in the pres-
ence of PoyD (left and right panels respectively). Amino acids
were analyzed after acid hydrolysis and derivatization with Nꢀαꢀ
(2,4ꢀdinitroꢀ5ꢀfluorophenyl)ꢀLꢀvalinamide (LꢀFDVA) and their
retention times compared with authentic standards. (d) HPLC
analysis of SAM incubated with PoyD. Traces indicate incubaꢀ
tion under anaerobic conditions in the presence of sodium dithioꢀ
nite at t=0 and after 90 min. As shown, during incubation Sꢀ
adenosyl Lꢀmethionine (SAM) is cleaved in 5'ꢀdeoxyadenosine
(5'ꢀdA). (e) LC-MS analysis of 5'-deoxyadenosine (5'-dA)
produced by PoyD. (f) Activity of PoyD toward peptide 1.
HPLC Analyses were performed at t=0, 30, 60, 90 and 120 min
(lower to upper traces respectively). See Supplementary Inforꢀ
mation for experimental conditions.
In order to determine the influence of the leader peptide on
the activity and specificity of PoyD, we synthesized a peptide
containing the residues +3 to +10 (Peptide 7, Figure 3d).
With this peptide, devoid of residues from the leader peptide,
PoyD catalyzed the formation of five peptides (Peptides 8, 9,
10, 11 & 12) (Figure 3d). LCꢀMS/MS analysis showed that
the main products formed were two peptides with three epiꢀ
merized residues (i.e. Valꢀ5, Valꢀ7 and Valꢀ9 or Valꢀ6, Ala8
and Valꢀ10, Peptides 11 & 12, respectively), two peptides
with two epimerized residues (i.e. Valꢀ7 and Valꢀ9 or Alaꢀ8
and Valꢀ10, Peptides 10 & 9, respectively) and a very low
amount of a monoꢀepimerized peptide (Peptide 8) (see Sup-
plementary Figures S8-S13 & Supplementary Tables S7-
S12).
In each condition, PoyD catalyzed the reducing cleavage of
SAM into 5ʹꢀdA (Figure 2d & e; [M+H]+= 252.1) demonstratꢀ
ing its activity as a radical SAM enzyme. Incubation of PoyD
with peptide 1 led to the formation of three peptides (Peptides
4, 5 & 6, eluting at 30.1, 30.4 & 30.9 min, respectively) (Fig-
ure 2f). Mass spectrometry analysis revealed no mass differꢀ
ence between peptide 1 and the products formed (Supple-
mentary Figure S2). In order to ascertain the nature of the
modification and to identify the modified residues, we perꢀ
formed the reaction in deuterated buffer. Indeed, vivo23ꢀ24 and
in vitro5 investigations of radical SAM epimerases have shown
that they introduce solvent derived Hꢀatoms into their prodꢀ
ucts. Under these conditions, the peptides produced had their
masses shifted from [M+2H]2+= 969.6 to [M+2H]2+= 970.6 for
peptides 4 & 5 and to [M+2H]2+= 971.1 for peptide 6 (Figure
3a).
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the activity of PoyD is largely independent of the leader pepꢀ
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tide. Of note, PoyD produced several peptides with epimerizaꢀ
tion located on the last residue (i.e. peptides 4, 9 & 12), in
sharp contrast with a recent in vivo study which suggested that
PoyD cannot modify the last residue of truncated PoyA pepꢀ
tides23. Finally, because the postꢀtranslational modifications
accumulated at the Cꢀterminal end of the various peptides
assayed, our results were consistent, as recently suggested by
in vivo experiments, that PoyD has a directional activity from
the Cꢀterminal toward the Nꢀterminal end of the peptide.
To date, several radical SAM enzymes such as AlbA8ꢀ9 and
YydG5 have been shown to introduce multiple postꢀ
translational modifications in their substrate, in vitro. Howevꢀ
er, there is no evidence that one molecule of enzyme is reꢀ
sponsible for the insertion of several postꢀtranslational modifiꢀ
cations on one molecule of substrate. Only for the radical
SAM enzyme lipoyl synthase, it has been shown that one
molecule of enzyme introduces sequentially two modifications
in its substrate (i.e. insertion of two sulfur atoms into its fatty
acyl substrate)25. However, the recently solved structure of
lipoyl synthase has shown that the incorporation of the two
sulfur atoms is part of the same catalytic event26 and does not
imply enzyme motion. The production by PoyD of peptides
containing several epimerized residues indicated either the
combined action of several enzymes on a same peptide backꢀ
bone or a processive activity of PoyD.
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Mechanistic investigation of PoyD. Having developed an
in vitro assay for PoyD, we were able to interrogate its mechaꢀ
nism. MS analysis of the epimerized amino acid residues (i.e.
DꢀVal &
DꢀAla) produced by PoyD in deuterated buffer
showed a mass shift of +1 Da compared to their
Lꢀconfigured
Figure 3 – Characterization of the products formed in vitro by
PoyD. (a) LC-MS/MS analysis of peptide 1 incubated with
PoyD. Upper trace peptide 1. Lower trace, peptide 1 incubated
with PoyD for 2 hours, in deuterated buffer. See Supplementary
Information for experimental conditions. Numbers refer to the
corresponding peptides. (b) Sequence of the different products
formed by PoyD in vitro after incubation with peptide 1 or
peptide 2. See Figure 1 for the amino acids residues correspondꢀ
ing to R & R’ and Figures S3ꢀ4 and S6 and Supplementary Tables
S1ꢀS6 for complete peptide assignment. (c) LC-MS/MS analysis
counterparts (Figure 4a & Supplementary Figure S5). This
result, in line with in vivo studies23ꢀ24, confirmed that one
solventꢀderived deuterium atom was incorporated during caꢀ
talysis. However, MS analysis of the 5ʹꢀdA produced ([M+H]+:
252.1) showed no deuterium incorporation consistent with
PoyD abstracting a substrate nonꢀexchangeable Hꢀatom (Fig-
ure 4b). To further validate this conclusion, we incubated
PoyD in deuterated buffer but omitted the peptide substrate.
Under these conditions, the molecular weight of 5ʹꢀdA shifted
to [M+H]+: 253.1 (Figure 4b, middle panel) indicating that in
absence of its substrate, PoyD still generated 5ʹꢀdA• but that
this latter reacted with buffer components (Figure 4b).
of the L-/D-Val content in peptide 1 and the products: peptides
4, 5 & 6 obtained after incubation with PoyD. Amino acids
were analyzed after hydrolysis and derivatization by LꢀFDVA and
detected by LCꢀMS after ion current extraction in MS/MS experꢀ
iments using the transition 398>352 for ValꢀFDVA derivatives.
Numbers indicate peptides analyzed. (d) LC-MS/MS analysis of
peptide 7 incubated with PoyD. Analysis was performed at t=0
and after 2 hours incubation, upper and lower traces, respectively.
Sequences of the peptides produced are indicated. See Suppleꢀ
mentary Figures S8ꢀ13 and Supplementary Tables S7ꢀS12 for full
peptide assignment. Numbers refer to the corresponding peptides.
Kinetic experiments performed with peptide 1 showed that
formation of peptide 4 stopped after 90 minutes (Figure 4c)
while peptide 6 was produced over three hours with an estiꢀ
mated kcat (per epimerization) of 0.02 minꢀ1 and 0.03 minꢀ1,
respectively (Figure 4c). Thus, peptide 6, with the unnatural
epimerization pattern, was the most efficiently produced pepꢀ
tide, in vitro. Interestingly, after an initial accumulation, pep-
tide 5 tended to disappear while production of peptide 6 still
proceeded. This result suggested that peptide 5 could serve as
substrate for PoyD and further converted into peptide 6. Since
peptides 4 and 5 have two modifications and peptide 6 has
three modifications, production of 5ʹꢀdA (~1100 µM) and the
three epimerized peptides (peptide 4 (157 µM), peptide 5
(23µM) and peptide 6 (248 µM)) indicated a good correlation
between epimerization events and SAM consumption (Figure
4c & d). In addition, LCꢀMS analysis of 5ʹꢀdA produced overꢀ
time in deuterated buffer exhibited no deuterium incorporaꢀ
tion. Only when the substrate became limiting (after one
hour), we monitored <10% deuterium incorporation in 5ʹꢀdA
Thus, this short substrate, despite lacking residues from the
leader peptide, recapitulated the different epimerization patꢀ
terns obtained with peptides 1 & 2.
Collectively, these data showed that subtle variations in the
sequences of the substrates led to the formation of peptides
with the epimerization pattern found in polytheonamide A (i.e.
peptides 3, 4, 9 & 12) but also peptides with an unnatural
epimerization pattern (i.e. peptides 5, 6, 8, 10 & 11). Howevꢀ
er, PoyD always catalyzed epimerization of residues from the
core sequence at 1,3ꢀpositions but never of residues from the
leader sequence. In addition, these experiments support that
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(Figure 4b, lower trace) while no labeling was measured in
the remaining SAM. Altogether, these results were consistent
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with one molecule of SAM being used by epimerization event.
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Identification of a potential critical H-atom donor. We
have recently discovered a peptidyl epimerase in Bacillus
subtilis. This enzyme called YydG possesses, in addition to
the radical SAM cluster, an additional [4Feꢀ4S] cluster5. This
auxiliary cluster has been proposed to assist radical quenching
during catalysis5. Interestingly, the only other radical SAM
epimerase characterized in vitro, NeoN27 which epimerizes the
Cꢀ5''' of neomycin, also contains an additional [4Feꢀ4S] clusꢀ
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ter27 in a SPASMꢀlike domain12, 28ꢀ29
.
Sequence analysis of PoyD revealed no obvious motif for
the coordination of an additional [4Feꢀ4S] cluster among the
10 cysteine residues present within the protein sequence. To
confirm this hypothesis, we replaced the cysteine residues of
the radical SAM motif (CxxxCxxC) by alanine residues and
probed for the presence of additional [4Feꢀ4S] clusters. After
purification and anaerobic reconstitution, the triple Cys→Ala
mutant (A3 mutant) exhibited a distinct UVꢀvisible spectrum
from the wildꢀtype enzyme (Figures 5a & b). Determination
of the iron content indicated that the A3 mutant contained 0.3±
0.2 mol of Fe per polypeptide consistent with its UVꢀvisible
spectrum showing the absence of ironꢀsulfur clusters. To assay
its activity, we coꢀexpressed in vivo the A3 mutant with PoyA.
As shown (Figure 5c), no epimerized residues could be identiꢀ
fied in PoyA supporting the critical role of the radical SAM
cluster for PoyD activity.
In several radical SAM enzymes such as spore photoproduct
lyase30ꢀ34, PolH35, NeoN27 or YydG5, it has been shown that a
cysteine residue is used as a critical Hꢀatom donor. However,
because of the lack of significant homology between PoyD
and these enzymes, we could not identify a putative Hꢀatom
donor. We thus aligned the sequence of PoyD with several
PoyDꢀhomologs recently identified24. In addition to the three
cysteine residues from the radical SAM motif, only one cysteꢀ
ine residue (Cys-372) was conserved among these enzymes
(Figure 5d). We further searched for homolgs in protein dataꢀ
bases and identified 67 homologs (sequence identity> 25%)
mostly in Proteobacteria. Sequences alignment confirmed that
beside the cysteines from the radical SAM motif, only one
cysteine residue (i.e. Cysꢀ372 in PoyD) was conserved among
these proteins (Supplementary Figure S14).
Figure 4 – Mechanistic and kinetic analysis of the reaction
catalyzed by PoyD. (a) MS/MS spectra of -Val (upper traces)
and -Val (lower traces) obtained after incubation of PoyD
with peptide 1 in deuterated buffer. Peptide products were
purified and after acid hydrolysis, amino acids were derivatized
L
D
with LꢀFDVA and analyzed by LCꢀMS/MS. See Supplementary
Information for experimental conditions. (b) MS spectra of 5'-dA
produced by PoyD. PoyD was incubated in deuterated buffer
under anaerobic and reducing conditions in the presence (upper
trace) or in the absence of peptide 1 (middle trace). The lower
trace shows MS spectra of 5'ꢀdA produced over time (from t=0 to
t=240 min) in the presence of peptide 1 in deuterated buffer.
Production of epimerized peptides (c) and 5'-dA (d) by PoyD.
PoyD was incubated in the presence of peptide 1 under anaerobic
conditions with sodium dithionite and SAM. Numbers refer to the
corresponding peptides formed.
To probe for the function of Cysꢀ372, we performed its
Cys→Ala replacement and coꢀexpressed the corresponding
mutant (C372A mutant) in vivo with PoyA. Interestingly, the
C372A mutant failed to epimerize PoyA (Figure 5c). Howevꢀ
er, analysis of the purified C372A mutant showed that its iron
content increased from 0.2± 0.1 to 3.5± 0.2 mol of Fe per
polypeptide after anaerobic reconstitution, similarly to the
wildꢀtype enzyme.
We further assayed the activity of the C372A and A3 muꢀ
tants against peptide 1, in vitro (Figure 5e). As shown, the A3
mutant had no enzymatic activity inꢀline with the in vivo exꢀ
periments. In contrast, the C372A mutant proved to produce a
novel peptide (peptide 13) eluting at 29.4 min and distinct
from peptides 4, 5 & 6, produced by the wildꢀtype enzyme.
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Page 6 of 11
in vitro30, 33, 37. Altogether, these results support that Cysꢀ372
1
2
3
4
fulfills an important function likely as a critical Hꢀatom donor.
However, we cannot rule out that other residues are involved
in this process notably tyrosine residues, as shown for carꢀ
bapenem synthase38.
Processivity of PoyD. The fact that the C372A mutant proꢀ
duced peptide with only one epimerized residue, while the
wildꢀtype enzyme systematically produced peptides with mulꢀ
tiple epimerizations, prompted us to assay the activity of PoyD
against the monoꢀepimerized peptide 13. Indeed, the failure of
the C372A mutant to catalyze multiple epimerizations sugꢀ
5
6
7
8
9
10
11
12
13
14
15
16
17
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20
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23
24
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26
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28
29
30
31
32
33
34
35
36
37
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41
42
43
44
45
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47
48
49
50
51
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53
54
55
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60
gested that only peptides containing
Lꢀamino acid residues
could serve as substrates for PoyD. We thus incubated peptide
1 with the C372A mutant and purified peptide 13 (Figure
6a). This peptide was then further incubated with the wildꢀ
type enzyme and the reaction analyzed by HPLC and LCꢀMS.
In contrast to peptide 1, incubation of peptide 13 with the
wildꢀtype enzyme led to the formation of only two peptides
(peptides 5 and 6). The implication of this result is twofold: It
demonstrates that epimerized peptides are substrates for PoyD
and, more importantly, that the first epimerization event
guides and restricts the following epimerization events in
order to preserve the strict 1,3ꢀpattern of epimerization.
Figure 5 – Identification of a potential H-atom donor in PoyD.
(a) Gel electrophoresis analysis of the A3 and C372A mutants
expressed in E. coli. (b) UV-visible spectrum of the A3 and
C372A mutants before (dotted line) and after anaerobic re-
constitution (plain line). (c) HPLC analysis of the amino acid
content of PoyA after its in vivo expression in E. coli with the
A3 or the C372A mutants (left and right panels, respectively).
Amino acids were analyzed after acid hydrolysis and derivatizaꢀ
To try to discern between processivity and cooperativity, we
further performed kinetic experiments in the presence of an
equal amount of wildꢀtype (peptide 1) and monoꢀepimerized
peptide (peptide 13). As shown, peptide 13 was converted
threeꢀtimes faster than peptide 1 (Figure 6b). In agreement
with this result, peptide 5, which was a minor species when
PoyD was incubated with peptide 1 alone (Figure 4c), was
the dominant product formed during the first 30 minutes of the
reaction (Figure 6c). As the reaction proceeded, peptide 5
tion with LꢀFDVA and their retention times compared with auꢀ
thentic standards. See Supplementary Information for experiꢀ
mental conditions. (d) Sequence alignment between PoyD and
other proteusin epimerases OspD, AvpD and PlpD. Strictly
conserved residues are highlighted in grey or red (cysteine resiꢀ
dues). Numbers refers to amino acid residues location in the
respective sequences. (e) HPLC analysis of peptide 1 incubated
in the presence of the A3 or C372A mutant. Upper trace: HPLC
analysis of peptide 1 at t=0. Middle trace: HPLC analysis of
peptide 1 after 120 min incubation with the A3 mutant. Lower
trace: HPLC analysis of peptide 1 after 120 min incubation with
the C372A mutant. The sequence of the product formed by the
C372A mutant is indicated. See Supplementary Figure S15 and
Supplementary Table S13 for full assignment. (f) LC-MS/MS
was then further converted into peptide 6 containing three
Dꢀ
amino acid residues (Figure 6c). These results demonstrate
that PoyD has a better activity on a peptide containing an
epimerized residue rather than on a peptide containing only Lꢀ
amino acid residues. However, in contrast to peptide 1 which
was converted into peptides with different epimerization patꢀ
terns, peptide 13 was converted only into peptide 5 and ultiꢀ
mately peptide 6 (Figure 6). This suggests that binding and
positioning of the substrate is determined, in part, by the presꢀ
ence of epimerized residues.
analysis of the L-/D-Val content of peptides 1 & 13. Upper trace
corresponds to peptide 1 and lower trace to peptide 13 produced
by the C372A mutant. LC MS/MS experiments were performed
using the transition 412>366.
LCꢀMS/MS analysis of this novel peptide showed the penulꢀ
timate valine residue, Valꢀ9, to be epimerized (Supplemen-
tary Figure S15). This result was further confirmed by amino
acid analysis which confirmed the presence of
Dꢀvaline in
peptide 13 with a ꢀVal/ ꢀVal ratio of ~20%, consistent with
D
L
the modification of one valine residue out of 5 (Figure 5f).
Thus, contrary to in vivo conditions, in vitro experiments
showed that the C372A mutant is able to catalyze peptide
epimerization.
Such apparent discrepancies, between in vivo and in vitro
activities, have been reported during the investigation of anꢀ
other radical SAM enzyme, the spore photoproduct lyase36, for
which mutation of the Hꢀatom donor (i.e. Cysꢀ141)30, 32ꢀ33 has
been shown to impair the DNA repair activity in spores but
not the ability of the enzyme to repair the spore photoproduct
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Journal of the American Chemical Society
enzyme of unknown function from Bacillus subtilis, we
demonstrated this enzyme to be a peptidyl epimerase5 convertꢀ
ing ꢀVal and ꢀIle into their epimers, during the biosynthesis
of the soꢀcalled epipeptides2, 5
1
2
3
L
L
.
4
5
6
7
8
9
Interestingly, despite being active on similar hydrophobic
amino acid residues, YydG is unrelated, at the sequence level,
to PoyD. Notably, YydG is devoid of the RiPP precursor pepꢀ
tide recognition element (i.e. RRE or PqqDꢀlike domain)42,
characteristic of PoyD and many RiPP modifying enzymes2. In
addition, our study shows that PoyD, in contrast to YydG,
contains only one [4Feꢀ4S] cluster.
10
11
12
13
14
15
16
17
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21
22
23
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25
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30
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34
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43
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46
47
48
49
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Epimerized peptides produced in vitro contained modificaꢀ
tions only in the Cꢀterminal region strongly supporting a Cꢀtoꢀ
N directionality for the enzyme, as recently suggested by in
vivo experiments. Interestingly, the recent in vitro study of
PoyC has demonstrated that it catalyzes methyl transfer to the
Cꢀterminal end of a synthetic peptide15. We can thus speculate
that PoyC, like PoyD, introduces postꢀtranslational modificaꢀ
tions with a similar CꢀtoꢀN directionality. Definitive proofs of
the directionality of PoyD came from the investigation of the
C372A mutant. Indeed, this mutant produced only a monoꢀ
epimerized peptide. The implications here are twofold: first, it
unveiled the initiation site of the peptide modification and
second, it suggests a processive mode of action of the enzyme.
Indeed, we did not evidence the production of other monoꢀ
epimerized products (i.e. peptides epimerized on other resiꢀ
dues) or the formation of peptides with several epimerizations,
as expected in case of the action of several molecules of enꢀ
zyme.
Interestingly, contrary to recent in vivo studies23, 43, peptides
produced in vitro by PoyD contained either the same epimerꢀ
ized residues than the ones found in polytheonamide A (i.e.
natural pattern of epimerization: peptides 3, 4, 9 & 12) or
unnatural epimerizations involving amino acid residues not
epimerized in polytheonamide A (i.e. peptides 5, 6, 8, 10 &
11).
Figure 6 – (a) HPLC analysis of peptide 1 after incubation
with the C372A mutant and wild-type PoyD. Peptide 1 was
incubated with the C372A mutant and analyzed at t=0 (upper blue
trace) and t=120 (red trace). After purification, peptide 13 was
incubated with PoyD and analyzed by HPLC at t=0 (green trace)
and t=120 min (purple lower trace). Numbers refer to the correꢀ
sponding peptides. (b) Consumption of peptides 1 and 13 dur-
ing incubation with PoyD. (c) Production of epimerized pep-
tides by PoyD. PoyD was incubated in the presence of peptide 1
under anaerobic conditions with sodium dithionite and SAM.
Numbers refer to the corresponding peptides formed.
Of note, we were able to obtain these epimerization patterns
using either peptides containing a portion of the leader peptide
(i.e. peptides 1 & 2) or peptides containing only residues from
the core sequence (i.e. peptide 7). Hence, the activity of PoyD
is largely independent of the leader peptide, as shown for other
radical SAM enzymes catalyzing peptide postꢀtranslational
modifications such as YydG5, AlbA9 and PoyC15, but in conꢀ
Interestingly, the transient accumulation of peptide 5, in the
range of the enzyme concentration (~150µM), is consistent
with at least a partially processivity of PoyD, as recently
shown for lanthipeptide synthetases39ꢀ40. Further studies will
be required to definitively address this question.
trast to other enzymes such as the KW_cyclase10, 12
.
Based on these results, we can propose the first mechanism
for PoyD and proteusin epimerases in general. Following the
reductive cleavage of SAM, PoyD generates 5ʹꢀdA• which
abstracts a substrate Cα Hꢀatom leading to the formation of 5ʹꢀ
dA and a carbonꢀcentered radical (Figure 7). After the loss of
the stereochemistry, one solvent exchangeable Hꢀatom is
transferred from an Hꢀatom donor (likely Cysꢀ372) to the
radical intermediate to produce an epimerized amino acid
residue and a thiyl radical on the protein.
DISCUSSION
Epimerization reactions were predicted to be catalyzed by
radical SAM enzymes more than a decade ago, consecutively
to the investigation of the avilamycin A biosynthetic
pathway41. In vivo studies have shown that a radical SAM
enzyme, AviX12, was responsible for a critical Cꢀ2 epimerizaꢀ
tion of a glucose moiety, essential to obtain the active form of
this antibiotic. Similarly, radical SAM epimerases have been
identified in the biosynthetic pathways of several RiPPs inꢀ
cluding the bacterial toxin polytheonamide A17, 23. However, it
is only recently that mechanistic insights have been gained on
these novel enzymes. The first radical SAM epimerase characꢀ
terized at the biochemical level, was the carbohydrate epiꢀ
merase NeoN which converts neomycin C into neomycin B27.
More recently, while investigating YydG, a radical SAM
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on ice in buffer A (Tris 50 mM, KCl 300 mM, pH 8) suppleꢀ
1
2
3
4
5
6
7
8
mented with protease inhibitor cocktail (EDTAꢀfree), 1%
Triton 100X and 0.2% 2ꢀmercaptoethanol. Cells debris were
removed by centrifugation at 45,000 x g for 1.5 hours and the
protein supernatant was loaded onto a Streptactin high capaciꢀ
ty gel (IBA) previously equilibrated with buffer A. The gel
was washed with 5 column volumes of buffer A and the PoyD
protein was eluted with 6 mL of buffer A containing desthioꢀ
biotine (3 mM) and dithiotreitol (DTT, 3 mM). The purified
protein was then concentrated with Amicon concentrator (moꢀ
lecular cutꢀoff of 10 kDa), aliquoted and stored at ꢀ80°C and
the protein purity was assayed on a 12% SDS–PAGE.
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17
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36
37
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60
Production of the PoyD mutant proteins. The pASK17+ꢀ
StrepꢀtagꢀPoyD plasmid served as template for siteꢀdirected
mutagenesis using this pair of primers:5′-ACA ACC AGC
GCT CTG ACC GGC-3′ /5′-GCC GGT CAG AGC GCT GGT
TGT-3′ to introduce an alanine at position 372. The triple
mutant C149A/C153A/C156A was obtained by two siteꢀ
directed mutagenesis. First, the C149A mutant was obtained
using the pASK17+ꢀStrepꢀtagꢀPoyD plasmid as template and
the primers: 5’ꢀACC CGT GGT GCT AGC GTT AAAꢀ3’ and
5’ꢀTTT AAC GCT AGC ACC ACG GGTꢀ3. The plasmid
pASK17+ꢀStrepꢀtagꢀPoyDꢀC149A was then used as DNA temꢀ
plate to introduce alanine mutations at positions 153 and 156
using the primers 5’ꢀCGT GGT GCT AGC GTT AAA GCT
TGG TTT GCT GCA CTGꢀ3’ and 5’ꢀCAG TGC AGC AAA
CCA AGC TTT AAC GCT AGC ACC ACG ꢀ3’. Clones were
selected on LB agar plate containing ampicillin (100 µg/L)
and DNA sequencing was performed to check the sequence of
the mutants. The plasmid was then used to transform E. coli
BL21 (DE3) star cells for protein expression. The expression
and purification of all mutant proteins were conducted in
similar conditions to the WT protein.
Figure 7 – Proposed mechanism for the radical SAM peptide
epimerase PoyD. After the reducing SAM cleavage, PoyD generꢀ
ates a 5′-dA• which abstracts the amino acid Cα Hꢀatom. A
carbonꢀcentred radical is formed and quenched by the thiolate Hꢀ
atom of Cysꢀ372 leading to the formation of a Dꢀamino acid resiꢀ
due. Reduction of the thiyl radical is likely assisted by other amiꢀ
no acid residues from PoyD similarly to ribonucleotide reductase
or spore photoproduct lyase for the next catalytic cycle.
In the absence of an additional [4Feꢀ4S] cluster, the most
likely hypothesis is that Cysꢀ372 is regenerated by the reducꢀ
tion of the thiyl radical by another cysteine residue like in
ribonucleotide reductase44. However, we did not identify an
obvious candidate to fulfill this function. Therefore, other
residues such as a tyrosine residue32ꢀ34, 37 may also be involved
in the regeneration of Cysꢀ372, as suggested for the radical
SAM enzyme spore photoproduct lyase30, 36, 45
.
To conclude, our study establishes that PoyD constiꢀ
tutes a distinct group of peptidyl epimerases within the superꢀ
family of radical SAM enzymes. We also demonstrate here
that the pattern of epimerization is likely an intrinsic property
of the enzyme which always produces epimerized peptides on
the 1,3ꢀpositions. Surprisingly, the enzyme is able to recognize
a peptide already containing one epimerized residue and to
catalyze the next epimerization event, preserving the 1,3 epiꢀ
merization pattern. The function of the leader peptide, even if
not essential, is likely to guide the positioning of PoyA within
PoyD active site for the first epimerization event. Further
studies should allow to decipher precisely how PoyD interacts
with its substrate and what are the molecular determinants of
its apparent processivity.
Enzyme reconstitution. Reconstitution of the [4Feꢀ4S]
cluster of PoyD and mutants, was achieved in a glove box
under strictly anaerobic conditions (< 1 ppm O2). Typically,
enzymes were reduced with 3mM DTT for 15 min prior rapid
addition of 6 molar equivalent of ammonium iron sulfate
hexahydrate ((NH4)2Fe(SO4)2) and sodium sulfide (Na2S)
followed by overnight incubation at 4°C. Excess unbound iron
and sulfur was removed by desalting proteins on Sephadex
G25 column against buffer A. Proteins was concentrated with
Amicon concentrator and concentration measured on
Nanodrop by measuring the absorbance at 280 nm with an
extinction coefficient value of 57,870 Mꢀ1.cmꢀ1.
Enzyme assays. Enzyme assays were performed at 25°C
under strictly anaerobic conditions in buffer A. Deuterated
buffer was obtained by several cycles of freezeꢀdrying in D2O.
Otherwise stated, 150 µM PoyD protein (WT or mutants after
anaerobic reconstitution), 2 mM SAM and 330 µM substrate
were mixed and the reaction initiated by adding 6 mM sodium
dithionite (DTN). For enzyme kinetics reactions, 10 µl aliꢀ
quots were sampled overtime for analysis and analyzed by
HPLC.
MATERIALS AND METHODS
Expression and purification of PoyD. The PoyD gene was
optimized for expression in E. coli and synthesized by Life
Technologies. The synthesized PoyD gene was inserted beꢀ
+
tween the NdeI and XhoI restriction sites of a pASK17 plasꢀ
mid with a Strepꢀtag fusion. The plasmid was then used to
transform E. coli BL21 (DE3) star cells. An overnight culture
of a single colony of E. coli BL21 (DE3)/pASK17+ꢀStrepꢀtagꢀ
PoyD was used to inoculate 9 L of LB medium containing
ampicillin (100 µg/L). Cells growth was carried out at 37°C
and 180 rpm until the OD at 600ꢁnm reached ∼0.7. Protein
expression was performed by adding anhydrotetracycline (400
µM) and iron citrate (250 µM). Cells were harvested by cenꢀ
trifugation (5 000 x g for 15 minutes at 4°C) after an incubaꢀ
tion time of 20 hours at 21°C and disrupted by ultraꢀsonication
Amino-acids enantiomer analysis after L-FDVA derivat-
ization. After reaction with PoyD, peptides were purified by
HPLC and hydrolyzed in DCl (or HCl, 6N) under vacuum
conditions at 110°C for 18h. Samples were dried using a cenꢀ
trifugal vacuum concentrator and dissolved in 10 µL MilliQ
water. Reaction mixtures were incubated 1 hour at 42 °C after
addition of 10 µl NaHCO3 1M and 25 µL Nαꢀ(2,4ꢀdinitroꢀ5ꢀ
fluorophenyl)ꢀLꢀvalinamide (LꢀFDVA, suspended in 0.5%
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Journal of the American Chemical Society
ACKNOWLEDGMENT
acetone). The derivatization reaction was stopped by addition
1
of 10 µL of HCl 2N. The mixture was diluted 1/10 in 20%
acetonitrile containing 0.1% formic acid before analysis. A
similar protocol was used to analyze the amino acid content of
PoyA expressed alone or with PoyD and PoyD mutants.
This work was supported by European Research Council (ERC)
grant: Consolidator Grant 617053.
2
3
4
ABBREVIATIONS
HPLC analysis. An Agilent 1200 series infinity equipped
with a reversed phase column (LiChroCART RPꢀ18e 5 µm,
Merck Millipore) was used to perform HPLC analysis. Samꢀ
ples were diluted 10ꢀfold in 0.1% trifluoroacetic acid solution.
The column was equilibrated with solvents A (H2O, 0.1%
TFA) and the solvent B (80 % CH3CN, 19.9 % H2O, 0.1%
TFA) was applied as follow: 0ꢀ1 min: 0% B; 1ꢀ20 min: linear
gradient with 1.2% B and 20ꢀ40 min: linear gradient with 3%
B at a flow rate of 1 min/L. Samples were diluted 10ꢀfold
before analysis. Detection was performed with a diode array
detector at 257, 278 and 340 nm and by fluorescence (ex/em:
278/350 nm).
5
6
7
8
SAM, SꢀadenosylꢀLꢀmethionine; 5’ꢀdAdo•, 5’ꢀdeoxyadenosyl
radical. RiPP: Ribosomallyꢀsynthesized and postꢀtranslationallyꢀ
modified peptide.
REFERENCES
9
1.
Arnison, P. G.; Bibb, M. J.; Bierbaum, G.; Bowers, A. A.;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Bugni, T. S.; Bulaj, G.; Camarero, J. A.; Campopiano, D. J.; Challis,
G. L.; Clardy, J.; Cotter, P. D.; Craik, D. J.; Dawson, M.; Dittmann,
E.; Donadio, S.; Dorrestein, P. C.; Entian, K. D.; Fischbach, M. A.;
Garavelli, J. S.; Goransson, U.; Gruber, C. W.; Haft, D. H.;
Hemscheidt, T. K.; Hertweck, C.; Hill, C.; Horswill, A. R.; Jaspars,
M.; Kelly, W. L.; Klinman, J. P.; Kuipers, O. P.; Link, A. J.; Liu, W.;
Marahiel, M. A.; Mitchell, D. A.; Moll, G. N.; Moore, B. S.; Muller,
R.; Nair, S. K.; Nes, I. F.; Norris, G. E.; Olivera, B. M.; Onaka, H.;
Patchett, M. L.; Piel, J.; Reaney, M. J.; Rebuffat, S.; Ross, R. P.; Sahl,
H. G.; Schmidt, E. W.; Selsted, M. E.; Severinov, K.; Shen, B.;
Sivonen, K.; Smith, L.; Stein, T.; Sussmuth, R. D.; Tagg, J. R.; Tang,
G. L.; Truman, A. W.; Vederas, J. C.; Walsh, C. T.; Walton, J. D.;
Wenzel, S. C.; Willey, J. M.; van der Donk, W. A., Ribosomally
synthesized and postꢀtranslationally modified peptide natural
Liquid chromatography - mass spectrometry analysis.
Mass spectrometry analysis was performed using an LTQ
standard mass spectrometer (Thermo Fisher Scientific) couꢀ
pling to a nanoꢀHPLC system (Ultimate 3000, Dionex thermo
fisher Scientific) with a nanoꢀelectrospray source. Samples
were inject onto a Pepmap100 C18 column (0,075 X 150 mm,
100A, 3µm ; Dionex) or a Proswift RP4H polymeric nanoꢀ
column (0.1 X 250 mm, 1000A, Dionex) (for PoyA), at a flow
rate of 0.3 µl/min and 0.45µl/min, respectively. The following
buffer system was used: Buffer A: formic Acid 0.1% and
buffer B: 80% acetonitrile, 0.1%formic Acid. Several linear
gradients were used according the molecules to analyze: 25 to
75% buffer B for peptide analysis; 0 to 20% buffer B for 5’ꢀ
dA; 30ꢀ100% buffer B for LꢀFDVA derivatives analysis and 0
to 60 % buffer B for PoyA analysis. Mass detection was realꢀ
ized in positive enhanced resolution. The doubly charge ions
for peptides 1 to 13 were fragmented at 35% NCE and spectra
acquired in profile and enhanced resolution mode to locate
deuterium incorporation. For LꢀFDVA derivatives, we extractꢀ
ed the ion current corresponding to the most intense ion
daughter detected in MS/MS spectrum (respectively the transiꢀ
tion 398>352 and 370>324 for ValꢀFDVA and AlaꢀFDVA
were used).
products: overview and recommendations for
a
universal
nomenclature. Nat. Prod. Rep. 2013, 30, 108ꢀ60.
2.
Benjdia, A.; Balty, C.; Berteau, O., Radical SAM Enzymes
in the Biosynthesis of Ribosomally Synthesized and Postꢀ
translationally Modified Peptides (RiPPs). Front. Chem. 2017, 5, 87.
3.
human microbiota. Science 2015, 349, 1254766.
4. Benjdia, A.; Berteau, O., Sulfatases and radical SAM
Donia, M. S.; Fischbach, M. A., Small molecules from the
enzymes: emerging themes in glycosaminoglycan metabolism and the
human microbiota. Biochem. Soc. Trans. 2016, 44, 109ꢀ15.
5.
Benjdia, A.; Guillot, A.; Ruffie, P.; Leprince, J.; Berteau,
O., Postꢀtranslational modification of ribosomally synthesized
peptides by a radical SAM epimerase in Bacillus subtilis. Nat. Chem.
2017, 9, 698ꢀ707.
6.
Hetrick, K. J.; van der Donk, W. A., Ribosomally
synthesized and postꢀtranslationally modified peptide natural product
discovery in the genomic era. Curr. Opin. Chem. Biol. 2017, 38, 36ꢀ
44.
7.
Products, Tailored To Fit. Acc. Chem. Res. 2017, 50, 1577ꢀ1586.
8. Flühe, L. K., T. A.; Gattner, M. J.; Schäfer, A.; Burghaus,
Funk, M. A.; van der Donk, W. A., Ribosomal Natural
ASSOCIATED CONTENT
Supporting Information
O.; Linne, U.; Marahiel, M. A., The radical SAM enzyme AlbA
catalyzes thioether bond formation in subtilosin A. Nat. Chem. Biol.
2012, 8, 350ꢀ7.
Supporting Information document includes experimental proceꢀ
dures, Figures S1ꢀS14 and Table S1ꢀ13.
This material is available free of charge via the Internet at
http://pubs.acs.org.
9.
Benjdia, A.; Guillot, A.; Lefranc, B.; Vaudry, H.; Leprince,
J.; Berteau, O., Thioether bond formation by SPASM domain radical
SAM enzymes: Calpha Hꢀatom abstraction in subtilosin
A
biosynthesis. Chem. Commun. 2016, 52, 6249ꢀ52.
10.
Schramma, K. R.; Bushin, L. B.; Seyedsayamdost, M. R.,
AUTHOR INFORMATION
Corresponding Author
Structure and biosynthesis of a macrocyclic peptide containing an
unprecedented lysineꢀtoꢀtryptophan crosslink. Nat. Chem. 2015, 7,
431ꢀ7.
* Olivier.Berteau@inra.fr
11.
Barr, I.; Latham, J. A.; Iavarone, A. T.; Chantarojsiri, T.;
Hwang, J. D.; Klinman, J. P., Demonstration that the radical Sꢀ
adenosylmethionine (SAM) enzyme PqqE catalyzes de novo carbonꢀ
carbon crossꢀlinking within a peptide substrate PqqA in the presence
of the peptide chaperone PqqD. J. Biol. Chem. 2016, 291, 8877ꢀ84.
Author Contributions
+ A.P., A.B., A.G. and X.K. contributed equally.
All authors have given approval to the final version of the manuꢀ
script.
12.
Benjdia, A.; Decamps, L.; Guillot, A.; Kubiak, X.; Ruffie,
P.; Sandstrom, C.; Berteau, O., Insights into the catalysis of a lysineꢀ
tryptophan bond in bacterial peptides by a SPASM domain radical Sꢀ
adenosylmethionine (SAM) peptide cyclase. J. Biol. Chem. 2017,
292, 10835ꢀ10844.
FUNDING SOURCES
This work was supported by European Research Council (ERC)
grant: Consolidator Grant 617053 to O.B.
13.
Pierre, S.; Guillot, A.; Benjdia, A.; Sandstrom, C.;
Langella, P.; Berteau, O., Thiostrepton tryptophan methyltransferase
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Journal of the American Chemical Society
Page 10 of 11
expands the chemistry of radical SAM enzymes. Nat. Chem. Biol.
2012, 8, 957ꢀ9.
31.
Chandor, A.; Berteau, O.; Douki, T.; Gasparutto, D.;
Sanakis, Y.; OllagnierꢀdeꢀChoudens, S.; Atta, M.; Fontecave, M.,
Dinucleotide spore photoproduct, a minimal substrate of the DNA
repair spore photoproduct lyase enzyme from Bacillus subtilis. J.
Biol. Chem. 2006, 281, 26922ꢀ31.
1
2
3
4
5
6
7
8
14.
Benjdia, A.; Pierre, S.; Gherasim, C.; Guillot, A.; Carmona,
M.; Amara, P.; Banerjee, R.; Berteau, O., The thiostrepton A
tryptophan methyltransferase TsrM catalyses
dependent methyl transfer reaction. Nat. Commun. 2015, 6, 8377.
15. Parent, A.; Guillot, A.; Benjdia, A.; Chartier, G.; Leprince,
a cob(II)alaminꢀ
32.
Benjdia, A.; Heil, K.; Winkler, A.; Carell, T.; Schlichting,
I., Rescuing DNA repair activity by rewiring the Hꢀatom transfer
pathway in the radical SAM enzyme, spore photoproduct lyase.
Chem. Commun. 2014, 50, 14201ꢀ4.
J.; Berteau, O., The B12ꢀradical SAM enzyme PoyC catalyzes valine
Cꢀbetaꢀmethylation during polytheonamide biosynthesis. J. Am.
Chem. Soc. 2016, 138, 15515ꢀ15518.
33.
Benjdia, A.; Heil, K.; Barends, T. R.; Carell, T.;
16.
Huo, L.; Rachid, S.; Stadler, M.; Wenzel, S. C.; Muller, R.,
Schlichting, I., Structural insights into recognition and repair of UVꢀ
DNA damage by Spore Photoproduct Lyase, a radical SAM enzyme.
Nucleic Acids Res. 2012, 40, 9308ꢀ18.
9
Synthetic biotechnology to study and engineer ribosomal bottromycin
biosynthesis. Chem. Biol. 2012, 19, 1278ꢀ87.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17.
Freeman, M. F.; Gurgui, C.; Helf, M. J.; Morinaka, B. I.;
34.
Benjdia, A., DNA photolyases and SP lyase: structure and
Uria, A. R.; Oldham, N. J.; Sahl, H. G.; Matsunaga, S.; Piel, J.,
Metagenome mining reveals polytheonamides as posttranslationally
modified ribosomal peptides. Science 2012, 338, 387ꢀ90.
mechanism of lightꢀdependent and independent DNA lyases. Curr.
Opin. Struct. Biol. 2012, 22, 711ꢀ20.
35.
peptidylnucleoside biosynthesis by radical SAM enzymes. Nat. Chem.
Biol. 2016, 12, 905ꢀ907.
36.
enzyme spore photoproduct lyase: From biochemistry to structural
investigations. Photochem. Photobiol. 2017, 93, 67ꢀ77.
37.
Stoll, S.; Schlichting, I.; Li, L., A radical transfer pathway in spore
photoproduct lyase. Biochemistry 2013, 52, 3041ꢀ50.
38.
Rosenzweig, A. C.; Boal, A. K.; Krebs, C.; Bollinger, J. M., Jr.,
Mechanism of the C5 stereoinversion reaction in the biosynthesis of
carbapenem antibiotics. Science 2014, 343, 1140ꢀ4.
Lilla, E. A.; Yokoyama, K., Carbon extension in
18.
Vey, J. L.; Drennan, C. L., Structural insights into radical
generation by the radical SAM superfamily. Chem. Rev. 2011, 111,
2487ꢀ506.
19.
Berteau, O.; Benjdia, A., DNA repair by the radical SAM
Broderick, J. B.; Duffus, B. R.; Duschene, K. S.; Shepard,
E. M., Radical Sꢀadenosylmethionine enzymes. Chem. Rev. 2014,
114, 4229ꢀ317.
20.
Ortillo, D.; Broderick, J. B.; Hoffman, B. M., Electronꢀnuclear double
resonance spectroscopic evidence that Sꢀadenosylmethionine binds in
contact with the catalytically active [4Feꢀ4S](+) cluster of pyruvate
formateꢀlyase activating enzyme. J. Am. Chem. Soc. 2002, 124, 3143ꢀ
51.
Yang, L.; Nelson, R. S.; Benjdia, A.; Lin, G.; Telser, J.;
Walsby, C. J.; Hong, W.; Broderick, W. E.; Cheek, J.;
Chang, W. C.; Guo, Y.; Wang, C.; Butch, S. E.;
21.
Nicolet, Y.; Amara, P.; Mouesca, J. M.; FontecillaꢀCamps,
39.
Thibodeaux, C. J.; Ha, T.; van der Donk, W. A., A price to
J. C., Unexpected electron transfer mechanism upon AdoMet
cleavage in radical SAM proteins. Proc. Natl. Acad. Sci. U. S. A.
2009, 106, 14867ꢀ71.
pay for relaxed substrate specificity: a comparative kinetic analysis of
the class II lanthipeptide synthetases ProcM and HalM2. J. Am.
Chem. Soc. 2014, 136, 17513ꢀ29.
22.
Horitani, M.; Byer, A. S.; Shisler, K. A.; Chandra, T.;
40.
Miller, L. M.; Chatterjee, C.; van der Donk, W. A.;
Broderick, J. B.; Hoffman, B. M., Why Nature Uses Radical SAM
Enzymes so Widely: Electron Nuclear Double Resonance Studies of
Lysine 2,3ꢀAminomutase Show the 5'ꢀdAdo* "Free Radical" Is Never
Free. J. Am. Chem. Soc. 2015, 137, 7111ꢀ21.
Kelleher, N. L., The dehydratase activity of lacticin 481 synthetase is
highly processive. J. Am. Chem. Soc. 2006, 128, 1420ꢀ1.
41.
S.; Koslowski, T.; Friedrich, T.; Bechthold, A., The active
conformation of avilamycin A is conferred by AviX12, a radical
AdoMet enzyme. J. Biol. Chem. 2006, 281, 14756ꢀ63.
Boll, R.; Hofmann, C.; Heitmann, B.; Hauser, G.; Glaser,
23.
Freeman, M. F.; Helf, M. J.; Bhushan, A.; Morinaka, B. I.;
Piel, J., Seven enzymes create extraordinary molecular complexity in
an uncultivated bacterium. Nat. Chem. 2017, 9, 387ꢀ395.
24.
Kegler, C.; Freeman, M. F.; Bode, H. B.; Piel, J., Radical Sꢀadenosyl
methionine epimerases: regioselective introduction of diverse Dꢀ
amino acid patterns into peptide natural products. Angew. Chem., Int.
Ed. 2014, 53, 8503ꢀ7.
42.
Burkhart, B. J.; Hudson, G. A.; Dunbar, K. L.; Mitchell, D.
Morinaka, B. I.; Vagstad, A. L.; Helf, M. J.; Gugger, M.;
A., A prevalent peptideꢀbinding domain guides ribosomal natural
product biosynthesis. Nat. Chem. Biol. 2015, 11, 564ꢀ70.
43.
Piel, J., An Orthogonal D2 OꢀBased Induction System that Provides
Insights into dꢀAmino Acid Pattern Formation by Radical Sꢀ
Adenosylmethionine Peptide Epimerases. Angew. Chem., Int. Ed.
2017, 56, 762ꢀ766.
Morinaka, B. I.; Verest, M.; Freeman, M. F.; Gugger, M.;
25.
Cicchillo, R. M.; Booker, S. J., Mechanistic investigations
of lipoic acid biosynthesis in Escherichia coli: both sulfur atoms in
lipoic acid are contributed by the same lipoyl synthase polypeptide. J.
Am. Chem. Soc. 2005, 127, 2860ꢀ1.
44.
Stubbe, J.; Nocera, D. G.; Yee, C. S.; Chang, M. C.,
Radical initiation in the class I ribonucleotide reductase: longꢀrange
protonꢀcoupled electron transfer? Chem. Rev. 2003, 103, 2167ꢀ201.
45.
OllagnierꢀdeꢀChoudens, S.; Fontecave, M.; Carell, T., The spore
photoproduct lyase repairs the 5Sꢀ and not the 5Rꢀconfigured spore
photoproduct DNA lesion. Chem. Commun. 2006, 445ꢀ7.
26.
McLaughlin, M. I.; Lanz, N. D.; Goldman, P. J.; Lee, K.
H.; Booker, S. J.; Drennan, C. L., Crystallographic snapshots of sulfur
insertion by lipoyl synthase. Proc. Natl. Acad. Sci. U. S. A. 2016, 113,
9446ꢀ50.
Friedel, M. G.; Berteau, O.; Pieck, J. C.; Atta, M.;
27.
Kudo, F.; Hoshi, S.; Kawashima, T.; Kamachi, T.; Eguchi,
T., Characterization of a radical SꢀadenosylꢀLꢀmethionine epimerase,
NeoN, in the last step of neomycin B biosynthesis. J. Am. Chem. Soc.
2014, 136, 13909ꢀ15.
28.
Benjdia, A.; Subramanian, S.; Leprince, J.; Vaudry, H.;
Johnson, M. K.; Berteau, O., Anaerobic sulfataseꢀmaturating enzyme
ꢀ a mechanistic link with glycyl radicalꢀactivating enzymes? FEBS. J.
2010, 277, 1906ꢀ20.
29.
Grell, T. A.; Goldman, P. J.; Drennan, C. L., SPASM and
twitch domains in Sꢀadenosylmethionine (SAM) radical enzymes. J.
Biol. Chem. 2015, 290, 3964ꢀ71.
30.
OllagnierꢀdeꢀChoudens, S.; Fontecave, M.; Atta, M., DNA repair and
free radicals, new insights into the mechanism of spore photoproduct
lyase revealed by single amino acid substitution. J. Biol. Chem. 2008,
283, 36361ꢀ8.
ChandorꢀProust, A.; Berteau, O.; Douki, T.; Gasparutto, D.;
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