In Vitro Characterization of the Colibactin-Activating Peptidase ClbP Enables Development of a Fluorogenic Activity Probe

Article abstract of DOI:10.1021/acschembio.9b00069

The gut bacterial genotoxin colibactin is linked to the development of colorectal cancer. In the final stages of colibactin's biosynthesis, an inactive precursor (precolibactin) undergoes proteolytic cleavage by ClbP, an unusual inner-membrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studied in vitro and limited tools exist to measure its activity. Here, we describe the in vitro biochemical characterization of catalytically active, full-length ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactin-producing bacteria.

Full text of DOI:10.1021/acschembio.9b00069

Subscriber access provided by AUT Library
Letter
In vitro characterization of the colibactin-activating peptidase
ClbP enables development of a fluorogenic activity probe
Matthew R. Volpe, Matthew R. Wilson, Carolyn A. Brotherton,
Ethan S. Winter, Sheila E. Johnson, and Emily P. Balskus
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00069 • Publication Date (Web): 06 May 2019
Downloaded from http://pubs.acs.org on May 7, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
ACS Chemical Biology
1
2
3
4
5
6
7
8
9
In vitro characterization of the colibactin-activating peptidase ClbP
enables development of a fluorogenic activity probe
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
Matthew R. Volpe, Matthew R. Wilson†, Carolyn A. Brotherton‡, Ethan S. Winter§, Sheila E. Johnson∥,
and Emily P. Balskus*
Department of Chemistry & Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138,
United States
alkylation.7,8 This proposal has been supported by the recent
ABSTRACT: The gut bacterial genotoxin colibactin is linked to
identification of colibactin-derived DNA adducts.9,10 We further
the development of colorectal cancer. In the final stages of
describe the reported structures of candidate precolibactins in a
colibactin’s biosynthesis, an inactive precursor (precolibactin)
Supplementary Discussion section.
undergoes proteolytic cleavage by ClbP, an unusual inner-
Previous studies revealed that ClbP possesses three C-terminal
transmembrane (TM) helices anchored in the inner membrane as
well as a soluble periplasmic peptidase domain.11 Prior in vitro
studies of ClbP used the truncated peptidase domain (ClbPpep), but
this construct does not rescue genotoxicity in a clbP mutant and
cannot process precolibactin-like synthetic substrates in whole
cells.12,13 In fact, deletion of even a single TM helix blocks
genotoxicity completely.11 Challenges in obtaining the full-length
enzyme (ClbPFL) have hampered the discovery and
characterization of ClbP inhibitors, with previous efforts limited to
in silico screening with ClbPpep and indirect phenotypic
measurements of activity.14 We therefore began our work by
characterizing ClbPFL in vitro.
membrane-bound periplasmic peptidase, to generate the active
genotoxin. This enzyme presents an opportunity to monitor and
modulate colibactin biosynthesis, but its active form has not been
studied in vitro and limited tools exist to measure its activity. Here,
we describe the in vitro biochemical characterization of
catalytically active, full-length ClbP. We elucidate its substrate
preferences and use this information to develop a fluorogenic
activity probe. This tool will enable the discovery of ClbP
inhibitors and streamline identification of colibactin-producing
bacteria.
The trillions of microorganisms living on and in the human body,
known as the human microbiota, have been implicated in many
diseases, including cancer. In particular, commensal gut bacteria
harboring the pks island are linked to colorectal cancer (CRC).1–3
The pks island encodes a hybrid nonribosomal peptide synthetase-
polyketide synthase (NRPS-PKS) assembly line that synthesizes
the small molecule genotoxin colibactin.4 Prior work has shown
that pks+ E. coli induce DNA double-strand breaks in eukaryotic
cells and increase tumor load in mouse models of CRC in an
inflammation-dependent fashion.2,5,6 While pks+ organisms are
present in ~20% of healthy individuals, they are more prevalent in
patients with inflammatory bowel disease (40%) and CRC (55%–
67%).2,5 Our understanding of colibactin’s role in carcinogenesis is
limited, as the active molecule has never been isolated or
structurally characterized and cannot be readily detected. We
sought to develop a fluorogenic probe that would allow us to
measure the activity of the colibactin biosynthetic pathway in a
more direct manner than current sequencing-based methods, which
detect the pks genes. Such a tool could also accelerate the
identification of small molecules that can inhibit colibactin
production.
Figure 1. ClbP is an essential part of colibactin’s prodrug
resistance mechanism. Precolibactin is synthesized by the NRPS-
PKS assembly line and tailoring enzymes encoded by the pks island
before hydrolysis by ClbP in the periplasm. While previous work
has shown that iminium formation (red arrows) is only possible
after cleavage by ClbP, the timing of lactam formation (blue
arrows) has not been firmly established (see also Figure S1). ‘R’ =
undefined.
We cloned and overexpressed C-terminal His10-tagged ClbP
with its TM helices intact (ClbPFL-CHis10), as well as an inactive
mutant lacking the catalytic serine nucleophile (ClbPFL-S95A-
CHis10), in E. coli C41 (Figure S2). We obtained purified enzyme
by separating the cell components using ultracentrifugation,
solubilizing the membranes in an n-dodecyl-β-D-maltoside
(DDM)-containing buffer, and performing immobilized metal
affinity chromatography. Because the structure of precolibactin has
not yet been fully elucidated, we tested purified ClbPFL against a
variety of synthetic precolibactin analogues. We found that ClbPFL
cleaves isolated candidate precolibactins, such as 1,15 as well as the
simplified precolibactin mimic 2 rapidly in vitro, while ClbPFL-
S95A and ClbPpep showed no activity by LC-MS (Figure 2A). The
kinetic parameters for cleavage of 1 and 2 by ClbPFL were
comparable (Figure S3), indicating the importance of the prodrug
scaffold for recognition and catalysis. We chose substrate 2 for a
more detailed structure-activity-relationship (SAR) study.
We chose the colibactin biosynthetic enzyme ClbP as a target for
probe development. This periplasmic peptidase proteolytically
activates colibactin in the final stages of genotoxin assembly. It
removes an N-myristoyl-D-Asn ‘prodrug scaffold’ from an inactive
precursor, precolibactin (Figure 1). Based on the structures of
candidate precolibactins isolated from clbP deletion mutants
(Figure S1), it has been hypothesized that cleavage by ClbP triggers
the formation of an ,-unsaturated iminium-conjugated
cyclopropane which serves as an electrophilic warhead for DNA
We first varied the N-acyl substituent of the prodrug scaffold.
We expected that ClbP would accommodate changes at this
ACS Paragon Plus Environment

ACS Chemical Biology
Page 2 of 9
position since E. coli overexpressing ClbPFL can hydrolyze
efficiency to substrate 2 (kcat/Km = 5400 ± 1800 vs 4600 ± 1100 M-
1s-1, Table S1), we synthesized an analogous probe containing the
same modification (17, Figure 3) in hopes of improving probe
solubility and performance. 17 was cleaved rapidly by ClbPFL in
vitro and showed negligible background activity with the S95A
mutant and ClbPpep (Figure 3B). It also exhibited excellent stability
in DMSO stock solutions, in vitro assay conditions, and LB and
MEGA growth media. In an in vitro assay containing 50 µM 17,
cleavage by ClbPFL results in a >100-fold increase in fluorescence
relative to the control in less than 30 minutes (Figure 3B). Probe 17
also shows robust and consistent cleavage in a whole-cell assay
format with E. coli overexpressing ClbP (Figure 3C).
1
2
3
4
5
6
7
8
synthetic precolibactin mimics bearing shorter fatty acyl chains.13
Thus, we synthesized a series of precolibactin mimics with varied
N-acyl chain length, polarity, and steric bulk (Table 1, entries 3–6).
LC-MS assays confirmed that ClbPFL hydrolyzed all of these
substrates at the expected peptide linkage (Table 1, Figure S4). We
also determined kinetic parameters for substrates 2, 3, and 4 using
a fluorescence-based assay with o–phthaldialdehyde (OPA, Figure
S5). The higher kcat and lower Km values observed for more
hydrophobic, long-chain substrates suggests acyl chain length is an
important, though non-essential, recognition feature (Table S1).
These observations, together with the fact that ClbPpep cannot
process 2 but retains some weak activity toward smaller
substrates,12 suggest that the TM helices could play a role in
substrate binding by interacting with the hydrophobic acyl
substituent.
9
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
Figure 3. Fluorogenic probes monitor ClbP activity in whole cells
and in vitro. (a) Mechanism of activation of fluorogenic probes. (b)
17 is activated by ClbPFL but not ClbPpep or ClbPFL-S95A in vitro.
Points represent mean fluorescence of 6 reactions for each enzyme
± SD. (c) 17 can be activated by whole cells. Points represent mean
fluorescence of 6 cultures of E. coli BL21 expressing each enzyme
± SD.
We next tested the ability of ClbPFL to hydrolyze substrates
bearing different amino acids within the prodrug scaffold (Table 1,
Entries 7–12). When ClbPFL was incubated with substrates
containing L-Asn, D-Gln, or D-Ala (8–10), no cleavage products
were detected by LC-MS (Figure S6). The D-Asp containing
substrate (7) was processed at low levels (>10% activity relative to
2), but we observed no hydrolysis of the corresponding D-Asp
methyl ester (11) and D-Asn dimethylamide (12) substrates (Figure
S6). These results indicate that this amino acid is a key recognition
motif. Two closely related peptidases, ZmaM and XcnG, also
hydrolyze substrates containing D-Asn at the same position in vivo,
suggesting that this feature is evolutionarily conserved.16,17
We next assessed whether our probe could detect ClbP activity
in wild-type pks+ strains. For this assay to provide a reliable read
out, it must be sensitive enough to be activated by native levels of
ClbP. Indeed, we reliably detected activation of probe 17 after
overnight incubation with the pks+ strains E. coli CCR20 and E.
coli Nissle 1917, as well as with a strain heterologously expressing
the pks island (BW25113 BACpks) (Figure 4A).
We next examined ClbP’s selectivity for the second amino acid
position of precolibactin mimics. Although all candidate
precolibactins characterized to date have L-alanine at this location,
ClbBNRPS, the module responsible for incorporating this building
block, accepts other amino acids in vitro.13 We incubated ClbPFL
with substrates 13–15 (Table 1) and detected cleavage of all
substrates by LC-MS (Figure S7). The observation that ClbPFL
accommodates variation at this position is in agreement with the
exceptionally large groove around the catalytic residues seen in
Finally, we used probe 17 to test the inhibition of ClbP by small
molecules. Boronic acids 18 and 19 were identified previously by
in silico docking with the ClbPpep crystal structure.14 These
molecules block the genotoxicity of pks+ E. coli toward mammalian
cells in tissue culture, but their effect on the catalytic activity of
ClbP has not been reported. When ClbPFL was incubated in vitro
with either 18 or 19 and probe 17, we observed a small decrease in
the fluorescent signal produced, but this change was not
statistically significant. This assay was also run under the same
conditions with half the amount of ClbPFL present. This control
showed approximately 50% activity, confirming that the assay is
sensitive to modest changes in the activity of the enzyme (Figure
4B). We also used a more sensitive LC-MS assay to evaluate the
inhibition of ClbPFL with substrate 2. In this format, we observed
weak but statistically significant inhibition of ClbPFL (10–15%
inhibition at 2 mM 18, Figure S9). The weakness of this effect
suggests that these molecules abrogate genotoxicity through other
cellular targets, and there remains a need for potent ClbP inhibitors.
As a first step toward identifying such molecules, we screened a
panel of known serine hydrolase inhibitors, including protease
inhibitors, -lactamase inhibitors, and lipase inhibitors, for ClbPFL
inhibition. None of the compounds tested showed inhibition at 2
mM concentration (Figure S10). The fact that even broad-spectrum
sulfonyl-fluoride and fluorophosphonate inhibitors cannot block
activity in vitro raises interesting questions about how ClbP’s
structure may regulate access to the active site.
crystal structure of ClbPpep.
11 The native precolibactin substrate is
likely a much larger molecule than 13–15, which may explain
ClbP’s promiscuity toward these substrates.
Figure 2. Characterization of ClbPFL activity in vitro. (a) LC-MS
detection of the prodrug scaffold ([M+H]+: 343.2597 m/z)
produced by ClbP-mediated hydrolysis of 100 µM 2 in vitro. Error
bars represent 1 standard deviation (SD), n=3. (b) SAR study of
a
ClbP substrates. based on detection of N-acyl-D-Asn cleavage
products by LC-MS. Figures S4-S7.
Using this information, we sought to develop a fluorogenic probe
for ClbP activity. Based on the key recognition elements we
identified, we designed a three-component probe that would
incorporate a large hydrophobic acyl substituent, the key D-Asn
residue, and a caged fluorophore connected to the prodrug scaffold
by a self-cleaving linker. This three-component approach has been
used successfully with more canonical types of peptidases.18–20 Our
initial target, 16, was accessed in 9 steps from commercially
available materials (see Supporting Information). Notably, the
activation of 16 is analogous to precolibactin activation: hydrolysis
by ClbP reveals a primary amine, which can undergo a rapid 5-exo-
trig cyclization to produce the active species (Figure 3A). While
ClbPFL processed 16 in vitro and in live E. coli, its low solubility
and membrane permeability likely led to poor performance in some
cell-based assays (Figure S8).
Figure 4. Probe 17 can identify pks+ organisms and assess
inhibition by small molecules. (a) Wild-type pks+ E. coli strains
were grown in MEGA medium under anaerobic conditions with
100 M 17 (Error bars are SD, n=3, Relative Fluorescence Units
(RFU) values measure whole culture). (b) Purified ClbPFL and the
indicated concentration of 18 or 19 were incubated for 1 hour and
fluorescence of each reaction was measured 25 minutes after
addition of 17 (Error bars are SD, treatment n=3, controls n = 9, 50
nM enzyme except where indicated.) Differences between
Because our SAR study indicated that ClbPFL hydrolyzed the 4-
phenylbutyryl-containing substrate
3 with similar catalytic
ACS Paragon Plus Environment

Page 3 of 9
ACS Chemical Biology
treatment groups and control were not significant (Student’s two-
tailed t test, P > 0.05).
In vitro activity and inhibition assays with fluorescent probes.
Putative inhibitors were prepared as 50 mM stocks in DMSO, while
probe 17 was prepared as a 10 mM stock in DMSO. ClbPFL-CHis10
was defrosted on ice from –80 °C stocks, diluted to 62.5 nM in
standard Tris assay buffer (50 mM Tris pH 8.0, 200 mM NaCl,
0.02% w/v DDM), and incubated with the appropriate
concentration of inhibitor or vehicle (DMSO) for 1 hour at ambient
temperature (n = 3 for each concentration). Separately, 250 µM 17
in Tris assay buffer was deposited in the wells of an opaque black
384-well flat-bottom plate (5 µL/well). Reactions were initiated by
transferring 20 µL of the enzyme/inhibitor or enzyme/DMSO mix
to the 384 well plate (final assay conditions: 0.1 µM enzyme, 50
µM probe 17, 0-2 mM inhibitor, 4.5% DMSO, 25 °C). Activity was
monitored by measuring the fluorescence of each well once per
minute for 4 hours using a Bio-Tek Synergy HTX multimode plate
reader (360/40 nm excitation filter, 440/20 nm emission filter).
Kinetic comparison of substrates 1 and 2 with ClbPFL (LC-
MS). In vitro reactions ClbPFL-CHis10 1 and 2 were conducted as
described above with assays for the SAR study with 50 nm
ClbPFLCHis10. Initial reaction rates were determined at various
concentrations of substrates by quenching 10 µL aliquots of the
reaction mixture in 90 µL of cold LC-MS grade methanol every
minute and analyzing these mixtures via LC-MS as described
above. Conversion was quantified using a standard curve of the
myristoyl-D-Asn cleavage product for both substrates (6
1
2
3
4
5
6
7
8
Despite over a decade of research, the colibactin genotoxin
remains elusive, and evidence of its carcinogenicity relies on
correlations and models that cannot fully recapitulate the
complexity of the gut microbiota. To better understand this intricate
pathway, we have characterized the essential colibactin-activating
peptidase ClbP in vitro and developed a fluorogenic probe for its
activity. Our SAR study provides a starting point for the rational
design of ClbP inhibitors, while our fluorogenic assay will allow
the use of high-throughput screening toward the same goal. Such
inhibitors would enable more detailed studies of colibactin’s
effects in physiologically-relevant animal models and in complex
microbiotas. The fact that our probe can detect ClbP activity in
wild-type pks+ strains also opens up the possibility of activity-
based diagnostics to detect these organisms.
9
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
ClbP is the most well-characterized enzyme in a larger family of
prodrug-activating peptidases. Though homologs ZmaM and XcnG
have been studied, ClbP is the only member of this family to be
purified and characterized in vitro. Qian and coworkers have shown
that additional peptidases that likely recognize N-acyl-D-Asn
motifs are encoded in cryptic gene clusters.21 Our fluorogenic
probe may be adaptable to these uncharacterized homologs,
enabling the direct monitoring of other secondary metabolite
pathways. Overall, this work shows how detailed characterization
of biosynthetic enzymes can enable the development of innovative
tools to interrogate natural products.
concentrations in triplicate). Rates were calculated at
7
concentrations for both substrates in 3 technical replicates and fit
to the Michaelis–Menten equation using the GraphPad Prism
software package.
Procedures and characterization data for all synthetic compounds
can be found in the Supporting Information. All other procedures,
including protocols for the purification of the enzymes discussed
and additional biochemical characterization, can also be found in
the Supporting Information.
METHODS
In vitro SAR study with substrates 2–15. Substrates were
prepared as 10 mM stock solutions in DMSO, then diluted to 120
µM in standard Tris assay buffer (50 mM Tris pH 8.0, 200 mM
NaCl, 0.02% w/v DDM). ClbPFL-CHis10 and ClbPFL-S95A-CHis10
were defrosted from –80 °C stocks on ice, then diluted to 0.6 µM
in Tris assay buffer. 25 µL of enzyme was deposited in individual
wells of a 96-well plate in triplicate. Reactions were initiated by the
addition of 125 µL of the appropriate substrate in Tris assay buffer
to a final concentration of 0.1 µM enzyme, 100 µM substrate, 1%
DMSO and incubated at 25 °C. A 20 µL sample of each reaction
mixture was immediately diluted into 180 µL of cold LC-MS-grade
methanol as the “t0” time point. Reactions were incubated at room
temperature for 5 hours, at which point another 20 µL of each
reaction mixture was diluted into 180 µL of cold LC-MS grade
methanol and mixed by pipetting (“t5”). 96-well plates containing
the methanol-quenched reactions were sealed with adhesive foil
and stored at –20 °C overnight. Plates were then centrifuged at
3,880 x g at 4 °C for 15 minutes and 20 µL of the supernatant was
diluted again into 180 µL of LC-MS-grade. Samples were analyzed
by LC-MS following the procedures outlined in the General
Materials and Methods (see Supporting Information), with the
exception of substrates 4, 5, and 6 for which the following
chromatography method was used: 0% solvent A in solvent B for
30 seconds, a gradient from 0% A to 50% A over 9.5 minutes,
followed by a gradient to 90% A over 2 minutes, holding at 90% A
for 3 minutes, a gradient back to 0% A over 1 minute, hold at 0%
A for 3 minutes (solvent A = acetonitrile + 0.1% formic acid;
solvent B = water +0.1% formic acid. Flow rate = 0.4 mL/minute,
injection volume = 10 µL). Relative activity was determined by
measuring the difference in Extracted Ion Chromatogram (EIC)
peak area for the expected mass of the prodrug scaffold fragment
(± 5 ppm) between the “t5” and “t0” samples and comparing this
value for each substrate to substrate 1 which was used as a control
in each sample group. For comparing substrates 2-6, a standard
curve for each substrate was used to quantify the molar
consumption of substrate. All substrates were assayed in triplicate.
ASSOCIATED CONTENT
Supporting Information
The Supporting information is available free of charge on the ACS
Publications website.
Detailed Materials and Methods; Supplementary Discussion;
Supplementary Figures S1-S9; Synthetic procedures and small
molecule characterization data.
AUTHOR INFORMATION
Corresponding Author
*balskus@chemistry.harvard.edu
Present Addresses
† Vertex Pharmaceuticals, 50 Northern Avenue, Boston, MA
02210, United States
‡Department of Microbiology, University of Washington School
of Medicine, 1705 NE Pacific Street, Seattle, WA 98195, United
States
∥Department of Biochemistry, University of Wisconsin-Madison,
433 Babcock Drive, Madison, WI 53706, United States
ORCID
Matthew R. Volpe: 0000-0002-0104-2929
Matthew R. Wilson: 0000-0001-8047-9469
Emily P. Balskus: 0000-0001-5985-5714
Notes
The authors declare the following competing financial interest: a
provisional patent has been filed on the fluorogenic probes
ACS Paragon Plus Environment

ACS Chemical Biology
Page 4 of 9
described here which lists M.R.V., M.R.W. and E.P.B. as co-
inventors (U.S. Provisional Application No.: 62/719,325).
(8) Balskus, E. P. (2015) Colibactin: understanding an elusive gut
bacterial genotoxin. Nat. Prod. Rep. 32, 1534–40.
1
2
3
4
5
6
7
8
§
(9) Wilson, M. R., Jiang, Y., Villalta, P. W., Stornetta, A.,
Boudreau, P. D., Carrá, A., Brennan, C. A., Chun, E., Ngo, L.,
Samson, L. D., Engelward, B. P., Garrett, W. S., Balbo, S., and
Balskus, E. P. (2019) The human gut bacterial genotoxin colibactin
alkylates DNA. Science. 363, eaar7785.
(10) Xue, M., Shine, E. E., Wang, W., Crawford, J. M., and Herzon,
S. B. (2018) Characterization of natural colibactin–nucleobase
adducts by tandem MS and isotopic labeling. Support for DNA
alkylation by cyclopropane ring opening. Biochemistry. 57, 6391-
6394.
(11) Cougnoux, A., Gibold, L., Robin, F., Dubois, D., Pradel, N.,
Darfeuille-Michaud, A., Dalmasso, G., Delmas, J., and Bonnet, R.
(2012) Analysis of structure-function relationships in the
colibactin-maturating enzyme ClbP. J. Mol. Biol. 424, 203–214.
(12) Dubois, D., Baron, O., Cougnoux, A., Delmas, J., Pradel, N.,
Boury, M., Bouchon, B., Bringer, M.-A., Nougayrede, J.-P.,
Oswald, E., and Bonnet, R. (2011) ClbP Is a Prototype of a
Peptidase Subgroup Involved in Biosynthesis of Nonribosomal
Peptides. J. Biol. Chem. 286, 35562–35570.
Deceased
ACKNOWLEDGMENT
The authors thank F. Prati for providing compound 18, R. Müller
and R. Bonnet for providing bacterial strains, Y. Jiang and P.
Boudreau for input on experimental design, J. Velilla Garcia and
R. Gaudet for plasmids and helpful discussion, C. Chittim and M.
Bollenbach for comments on the manuscript, and D. Kahne and
coworkers for use of equipment. The authors acknowledge
financial support from the National Institutes of Health
(R01CA208834) and the Damon Runyon-Rachleff Innovation
Award. M.R.W acknowledges support from the American Cancer
Society-New England Division Postdoctoral Fellowship (PF-16-
122-01-CDD). S.E.J acknowledges support from The Amgen
Scholars Program.
9
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
REFERENCES
(13) Brotherton, C. A., and Balskus, E. P. (2013) A Prodrug
Resistance Mechanism Is Involved in Colibactin Biosynthesis and
Cytotoxicity. J. Am. Chem. Soc. 135, 3359–3362.
(14) Cougnoux, A., Delmas, J., Gibold, L., Faïs, T., Romagnoli, C.,
Robin, F., Cuevas-Ramos, G., Oswald, E., Darfeuille-Michaud, A.,
Prati, F., Dalmasso, G., and Bonnet, R. (2016) Small-molecule
inhibitors prevent the genotoxic and protumoural effects induced
by colibactin-producing bacteria. Gut 65, 278–285.
(15) Zha, L., Jiang, Y., Henke, M. T., Wilson, M. R., Wang, J. X.,
Kelleher, N. L., and Balskus, E. P. (2017) Colibactin assembly line
enzymes use S-adenosylmethionine to build a cyclopropane ring.
Nat. Chem. Biol. 13, 1063–1065.
(16) Kevany, B. M., Rasko, D. A., and Thomas, M. G. (2009)
Characterization of the complete zwittermicin A biosynthesis gene
cluster from Bacillus cereus. Appl. Environ. Microbiol. 75, 1144–
1155.
(17) Reimer, D., Pos, K. M., Thines, M., Grün, P., and Bode, H. B.
(2011) A natural prodrug activation mechanism in nonribosomal
peptide synthesis. Nat. Chem. Biol. 7, 888–890.
(18) Prost, M., Canaple, L., Samarut, J., and Hasserodt, J. (2014)
Tagging live cells that express specific peptidase activity with
solid-state fluorescence. ChemBioChem 15, 1413–1417.
(19) Thorn-Seshold, O., Vargas-Sanchez, M., McKeon, S., and
Hasserodt, J. (2012) A robust, high-sensitivity stealth probe for
peptidases. Chem. Commun. (Camb). 48, 6253–5.
(20) Meyer, Y., Richard, J.-A., Delest, B., Noack, P., Renard, P.-
Y., and Romieu, A. (2010) A comparative study of the self-
immolation of para-aminobenzylalcohol and hemithioaminal-
based linkers in the context of protease-sensitive fluorogenic
probes. Org. Biomol. Chem. 8, 1777.
(21) Li, Y., Zhong, Z., Hou, P., Zhang, W., and Qian, P. (2018)
Resistance to nonribosomal peptide antibiotics mediated by d-
stereospecific peptidases. Nat. Chem. Biol. 14, 381–387.
(1) Buc, E., Dubois, D., Sauvanet, P., Raisch, J., Delmas, J.,
Darfeuille-Michaud, A., Pezet, D., and Bonnet, R. (2013) High
Prevalence of Mucosa-Associated E. coli Producing Cyclomodulin
and Genotoxin in Colon Cancer. PLoS One (Battista, J. R., Ed.) 8,
e56964.
(2) Arthur, J. C., Perez-Chanona, E., Muhlbauer, M., Tomkovich,
S., Uronis, J. M., Fan, T.-J., Campbell, B. J., Abujamel, T., Dogan,
B., Rogers, A. B., Rhodes, J. M., Stintzi, A., Simpson, K. W.,
Hansen, J. J., Keku, T. O., Fodor, A. A., and Jobin, C. (2012)
Intestinal Inflammation Targets Cancer-Inducing Activity of the
Microbiota. Science. 338, 120–123.
(3) Dejea, C. M., Fathi, P., Craig, J. M., Boleij, A., Taddese, R.,
Geis, A. L., Wu, X., Shields, C. E. D., Hechenbleikner, E. M.,
Huso, D. L., Anders, R. A., Giardiello, F. M., Wick, E. C., Wang,
H., Wu, S., Pardoll, D. M., Housseau, F., and Sears, C. L. (2018)
Patients with familial adenomatous polyposis harbor colonic
biofilms containing tumorigenic bacteria. Science. 359, 592–597.
(4) Nougayrede, J.-P., Homburg, S., Taieb, F., Boury, M.,
Brzuskiewicz, E., Gottschalk, G., Buchrieser, C., Hacker, J.,
Dobrindt, U., and Oswald, E. (2006) Escherichia coli Induces DNA
Double-Strand Breaks in Eukaryotic Cells. Science. 313, 848–851.
(5) Cougnoux, A., Dalmasso, G., Martinez, R., Buc, E., Delmas, J.,
Gibold, L., Sauvanet, P., Darcha, C., Déchelotte, P., Bonnet, M.,
Pezet, D., Wodrich, H., Darfeuille-Michaud, A., and Bonnet, R.
(2014) Bacterial genotoxin colibactin promotes colon tumour
growth by inducing a senescence-associated secretory phenotype.
Gut 1–11.
(6) Tomkovich, S., Yang, Y., Winglee, K., Gauthier, J., Mühlbauer,
M., Sun, X., Mohamadzadeh, M., Liu, X., Martin, P., Wang, G. P.,
Oswald, E., Fodor, A. A., and Jobin, C. (2017) Locoregional
Effects of Microbiota in
a Preclinical Model of Colon
Carcinogenesis. Cancer Res. 77, 2620–2632.
(7) Healy, A. R., Nikolayevskiy, H., Patel, J. R., Crawford, J. M.,
and Herzon, S. B. (2016) A Mechanistic Model for Colibactin-
Induced Genotoxicity. J. Am. Chem. Soc. 138, 15563–15570.
ACS Paragon Plus Environment

O
Cytosol
NH2
Page 5 of 9
ACS Chemical Biology
O
O
O
H
N
R
N
N
12
H
H
O
O
Precolibactin
NRPS/PKS assembly line
1
2
3
4
5
6
ClbM
O
Periplasm
R
H2N
NH
O
O
NH
O
R
ClbP
+
O
NH2
O
O
O
H
NH2
OH
N
R
O
7
NH
N
N
12
H
H
H
O
O
N
Colibactin
8
9
12
H
O
B
Prodrug scaffold
10
11
12
13
ACS Paragon Plus Environment
Genotoxicity

O
A
3
ACS Chemical Biology
Page 6 of 9
NH2
O
O
O
H
N
OH
N
N
H
H
O
12
2
1
O
1
1
O
2
3
4
5
6
7
NH2
O
O
H
N
0
N
O
H
12
O
2
0.1 µM ClbPFL
100 µM substrate
0.02% DDM
pH 8.0
B
8
O
R2
O
R2
H
9
OH
H2N
N
R1
N
1R01
11
N
R3
R3
+
H
H
O
O
Detect by LC-MS
12 R1
13
R2
R3
Accepted?a
2
C13H27
D-Asn
D-Asn
D-Asn
D-Asn
L-Ala-OMe Yes
L-Ala-OMe Yes
L-Ala-OMe Yes
L-Ala-OMe Yes
L-Ala-OMe Yes
L-Ala-OMe Trace
L-Ala-OMe No
L-Ala-OMe No
L-Ala-OMe No
14
3
(4-Ph)-C3H6
C3H7
15
4
16
5
CH3
17
6
(8-NH2)-C7H14 D-Asn
18
7
19
C13H27
C13H27
C13H27
D-Asp
L-Asn
D-Gln
D-Ala
20
8
21
9
22
10 C13H27
23
11 C13H27
D-Asp-4-OMe L-Ala-OMe No
D-Asn-4-NMe2 L-Ala-OMe No
24
12 C13H27
25
13 C13H27
D-Asn
Gly-OMe
Yes
Yes
26
ACS Paragon Plus Environment
27
14 C13H27
D-Asn
b-Ala-OMe
28
15 C13H27
D-Asn
L-Val-OMe Yes
29

E. coli expressing
O
A
ClbPFL
Page 7 of 9
ACS ChoermpuiricfieadlCBlbiPology
O
FL
NH2
O
H2N
H
N
O
O
N
O
N
R
N
O
O
O
1 O
2
3
O
O
O
NH2
OH
HN
O
16
R=
R
N
H
12
4
5
O
R=
17
N
H
O
O
O
6
7
B
C
83000
9
2000
1500
1000
500
2000
10
11
ClbPFL
FLS95A
ACS PaClbP
C
ralbPgon Plus Environment
112000
13
ClbPFL
pep
ClbPFLS95A
ClbPpepS95A
0
0
0
14
15
0
5
10
15
20
40
60
Time (minutes)
Time (hours)

500
A
CCR20
ACS Chemical Biology
Page 8 of 9
Nissle 1917
400
300
200
100
BW25113 BACpks
BW25113 BAC (empty)
Media
1
2
3
4
5
6
7
8
9
0
0
20
40
60
80
Time (hours)
10 800
B
HO
OH
B
11
12
13
14
15
16
17
18
19
20
21
22
600
400
NH2
18
O
O
B
200
0
NH
S
O
O
ACS Paragon Plus Environment
A
Cl
P
P
17
17
17
18
18
18
5
b
l
b
l
C
C
19
S9
2 mM1 mM
0.5 mM
2 mM 1 mM
0.5 mM
ClbP
50 nM

Precolibactin
Active colibactin
genotoxin
O
R
O
R
H
Page 9 of 9
ACS Chemical Biology
H2N
N
Acyl-D-Asn
NH
NH
O
NH
O
R
pks+ gut bacteria
1
2
3
4
5
6
7
NH
O
ClbP
Rational design
O
O
O
ACS Paragon Plus Environment
O
O
H
N
H2N
N
O
O
O
N
O
O
O
Acyl-D-Asn
Fluorescent
Signal
Activity probe