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Characterization of natural colibactin–nucleobase adducts by tandem MS and isotopic labeling. Support for DNA alkylation by cyclopropane ring opening. Mengzhao Xue, Emilee E. Shine, Weiwei Wang, Jason M. Crawford, and Seth B Herzon Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01023 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 27, 2018
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Biochemistry
Characterization of natural colibactin–nucleobase adducts by tandem MS and isotopic labeling. Support for DNA alkylation by cyclopropane ring opening. Mengzhao Xue,a Emilee Shine,b,c Weiwei Wang,d,e Jason M. Crawford,a,b,c* and Seth B. Herzona,f* a
Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States. Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States. c Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut 06536, United States. d Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, PO Box 208114, New Haven, CT, 06520. e W.M. Keck Biotechnology Resource Laboratory, Yale University School of Medicine, 300 George Street, New Haven, CT 06510. f Department of Pharmacology, Yale School of Medicine, New Haven, Connecticut 06520, United States. b
Supporting Information Placeholder ABSTRACT: Colibactins are genotoxic secondary metab-
olites whose biosynthesis is encoded in the clb gene cluster harbored by certain strains of gut commensal E. coli. Using synthetic colibactin analogs, we previously provided evidence that colibactins alkylate DNA by nucleotide addition to an electrophilic cyclopropane intermediate. However, natural colibactin–nucleobase adducts have not been identified, to our knowledge. Here we present the first identification of such adducts, derived from treatment of pUC19 DNA with clb+ E. coli. Previous biosynthetic studies established cysteine and methionine as building blocks in colibactin biosynthesis; accordingly, we used cysteine (DcysE) and methionine (DmetA) auxotrophic strains cultured in media supplemented with L-[U13 C]-cys or L-[U-13C]-met to facilitate the identification of nucleobases bound to colibactins. Using MS2 and MS3 analysis, in conjunction with the known oxidative instability of colibactin cyclopropane-opened products, we were able to characterize adenine adducts derived from cyclopropane ring-opening. This study provides the first reported detection of nucleobase adducts derived from clb+ E. coli and lends support to our earlier model suggesting DNA alkylation by nucleotide addition to an electrophilic cyclopropane.
Scheme 1. A. Structures of synthetic colibactins 1–3 and their reactivity toward DNA. B. Pathway for ClbS-mediated conversion of 4 to the hydroxyfuran 7. A. CH3
O N N H N H
O
N
N(CH 3) 2
N H
S
DNA CH3
O
N S
damages DNA
1
O N N H
CH3
N H
2
2
N CH3 H
O
NCH 3
N H
S O
N
N
S
N H
O
cross-links DNA
O
N
O
N
N
S
CH3
N(CH 3) 2
N H
S
unreactive toward DNA
3
B. CH3
O
ClbS
N N H
N
N
S
N H
O
O
CH 3
N H
NHCH 3 H 2O
4
N H
O
S
HO
5 O2
Colibactins are secondary metabolites encoded in a hybrid nonribosomal peptide synthetase–polyketide synthase (NRPS–PKS) gene cluster, referred to as clb, that is harbored by certain strains of gut commensal E. coli.1-5
O
CH 3
O
CH 3
O N H
N H
N S
O
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N OH H
7
N S
HO N
NHCH 3 O
6
N O H O H
Biochemistry 1 2 3 4 5 6 7 8 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
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1 2 3 4 5 6 7 The presence of the clb cluster is epidemiologically corΔcysE ΔmetA ladder vehicle cisplatin clb– clb+ related to colorectal cancer formation in humans, and studies suggests colibactins are genotoxic and cause tumors in mouse models. We previously provided evidence that colibactin genotoxicity derives in part from nucleotide addition to an electrophilic cyclopropane,6 a mechanism of DNA damage established for several classes of natural products.7, 8 Because natural colibactins have eluded isolation, this conclusion was based on studies of synthetic colibactin analogs such as 1–3 (Scheme 1A). First, linearized pBR322 plasmid DNA was extensively degraded following treatment with nanomolar concentrations of the synthetic colibactin analog 1, which bears the putative elecFigure 1. DNA cross-linking assays employing linearized Caption: DNA crosslinking assay employing linearized pUC19 DNA and E. coli or 100 µM cisplatin trophilic cyclopropane residue. Second, and consistent pUC19 DNA andConditions: E. coli variants. Cisplatin was used aspUC19 pos- DNA (31 µM in (positive control for crosslinking). a. cisplatin (100 µM), linearized with the cyclopropane behaving as a DNA electrophile, ⎻, BW25113 clb+, control. (Lane nob. treatment (Lane base pairs), 10itive mM pH 5 sodiumDNA citrateladder buffer, 4.5 hr at#1); 37 °C; BW25113 clb the dimer 2 was found to form DNA interstrand cross+#2); cisplatin + methionine (100or µM, Laneclb#3); clb– BW25113 coli pUC19 DNA ( auxotroph, JW3973-1 auxotroph,E. linearized links. Finally, the construct 3, which is identical to JW3582-2 1 save clb cysteine + 13C]-cys or L-[U-13C]-met for+the cys and met µM in base pairs), modified M9 media (containing L -[U(Lane #4); clb BW25113 E. coli (Lane #5); DcysE clb for conversion of the cyclopropane to a geminal-dimethyl auxotrophs, respectively), at 37 °C. The was isolated, purified, and by denaturin E.analyzed coli BW25113 4.5 E. hcoli (Lane #6);DNA DmetA clb+ BW25113 substituent, did not lead to detectable levels of DNAagarose dam- gel electrophoresis (90 V, 1.5 hr). (Lane #7). Conditions (Lanes #2 and #3): linearized pUC19 age (up to 500 µM concentration), as expected if the cyDNA (31 µM in base pairs), pH 5 sodium citrate buffer (10 clopropane were a DNA-reactive functionality. mM), 4.5 h, 37 °C. Conditions (Lanes #4 and #5): linearized Perhaps the strongest evidence implicating the cyclopUC19 DNA, M9 media, 4.5 h, 37 °C. Conditions (Lanes propane as a reactive locus underlying the genotoxicity of #6 and #7): linearized pUC19 DNA, modified M9 media clb+ E. coli was obtained through studies of the resistance (containing L-[U-13C]-cys or L-[U-13C]-met for cys and met auxotrophs, respectively), 4.5 h, 37 °C. DNA was isolated enzyme ClbS. ClbS is encoded in the clb cluster and was and analyzed by denaturing agarose gel electrophoresis (90 shown to be essential for bacterial viability.9 We demonV, 1.5 h). strated that purified ClbS cleaved the cyclopropane residue in the synthetic colibactin 4, ultimately resulting in facilitating their identification. clb– E. coli were used as formation of the 3-hydroxytetrahydrofuran 7 (Scheme a negative control. 1B).10 The formation of 7 was shown to proceed by ClbSLinearized pUC19 DNA and bacteria were incubated in catalyzed cyclopropane hydrolysis (4®5), aerobic oxidaM9 media for 4.5 h at 37 ºC. The bacteria were separated tion (5®6), and cyclization with concomitant reduction by centrifugation, and the DNA was isolated and analyzed of the alkyl hydrogen peroxide (6®7). This study estabby denaturing gel electrophoresis. As shown in Figure 1, lished that the bacteria evolved a mechanism to eliminate DNA was cross-linked when exposed to clb+, DcysE, or self-toxicity deriving from the reactivity of the cycloproDmetA E. coli, but not when exposed to clb– E. coli, as pane. expected. The cross-linked DNA was digested with the Despite these advances, the identification of natural Nucleoside Digestion Mix (New England Biolabs) and colibactin–nucleobase adducts has not been described, to analyzed by LC-MS/MS. our knowledge. In a recent study, clb+ E. coli were Prominent peaks at m/z = 522.1668, 538.1618, demonstrated to cross-link exogenous DNA.11 This sug540.1775, and 556.1722 (z = 1), and 261.5871, 269.5844, gested to us the possibility of characterizing natural coli270.5925, and 278.5899 (z = 2) were identified in DNA bactin–nucleobase adducts directly from bacterial cultreated with clb+ E. coli. These peaks were mass-shifted tures. To achieve this, here we conducted tandem MS by +3 or +4 units (z = 1) or +1.5 or +2 units (z = 2) in the analysis of the products formed after incubation of linearDcysE + L-[U-13C]-cys and DmetA + L-[U-13C]-met culized pUC19 plasmid DNA with clb+ E. coli BW25113. tures, respectively. The +3 or +1.5 mass shift in the prodWe conducted parallel assays using a cysteine auxotroph ucts derived from the DcysE culture indicates the presence (DcysE) and a methionine auxotroph (DmetA)12 cultured of only one thiazole ring. These observed masses fit the 13 13 in media supplemented with L-[U- C]-cys or L-[U- C]proposed ion structures 8–11 within 1.5 ppm of error (Figmet, respectively. Biochemical studies have established ure 2 and Table 1). that the thiazole and aminocyclopropane residues of coli13 The connectivity of 8–11 (in particular, the location of bactins are derived from cysteine and methionine via 13-15 the adenine base) was established by extensive MS2 and SAM, respectively. Thus, products derived from clb MS3 analysis in conjunction with the mass shifts in the metabolites were expected to be mass-shifted by 3 units auxotrophic strains anticipated based on the known label for each cysteine or 4 units for each methionine in these origins of the thiazole and cyclopropane residues.13, 14 auxotrophs,
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Biochemistry
CH3
CH3 O
N
N H O
N H
N S
OH
O
O
N H
Ad
N H
O
O
N
N H O
N
9 (m/z = 538.1818)
CH3
O
N
O
N H OH S
Ad
8 (m/z = 522.1668)
CH3
O
N
N S
OH
O
O
OH
N H
Ad•H+
N H OH S
O
N
O OH
Ad•H+
10 (m/z = 540.1775)
11 (m/z = 556.1722)
Figure 2. Proposed structures of colibactin–adenine adducts derived from incubation of pUC19 DNA with clb+ E. coli. Ad = adenine. Table 1. HRMS data of colibactin–nucleobase adducts.
strain
ion
z
exp.
theo.
error (ppm)
clb+
8
1
522.1668
522.1666
0.38
9
1
538.1618
538.1616
0.37
10
1
540.1775
540.1772
0.56
11
1
556.1722
556.1721
0.18
8
2
261.5871
261.5870
0.38
9
2
269.5844
269.5844
0.00
10
2
270.5925
270.5922
1.11
+
clb ΔcysE
11
2
278.5899
278.5897
0.72
13
C3-8
1
525.1765
525.1767
0.29
13
C3-9
1
541.1714
541.1717
0.46
C3-10 1
543.1874
543.1873
0.28
559.1822
559.1822
0.09
13
13
13
C3-8
2
263.0920
263.0920
0.10
13
C3-9
2
271.0897
271.0894
1.01
C3-10 2
272.0976
272.0972
1.38
280.0947
280.0947
0.09
13
13 +
clb ΔmetA
C3-11 1
C3-11 2
13
C4-8
1
526.1804
526.1800
0.76
13
C4-9
1
542.1745
542.1750
0.92
C4-10 1
544.1907
544.1906
0.18
560.1860
560.1855
0.89
13
13
C4-11 1
13
C4-8
2
263.5938
263.5937
0.38
13
C4-9
2
271.5914
271.5911
1.10
C4-10 2
272.5992
272.5989
1.10
280.5965
280.5964
0.36
13
13
C4-11 2
Thus, the daughter ions 15 and 19 were observed in the MS of 10 (Scheme 2). Ion 14 was observed as a daughter ion in the MS and in the MS2 spectra of 10, while ions 12 and 13 were only observed in the MS2 spectrum of 10. The thiazole 14 was mass-shifted by +3 units in the DcysE culture but did not change in the DmetA culture, consistent with the known biosynthesis of the thiazole residues from cysteine. The ions 15 and 19 were massshifted by +4 units in the DmetA culture but did not shift in the DcysE culture, consistent with the derivation of the cyclopropane from labeled methionine. The mass of adenine•H+ adducts were detected in the MS2 spectrum of unlabeled 10 and its cys and met-labeled isotopologs (error
