Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S-Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases

Article abstract of DOI:10.1021/jacs.5b05950

Identifying an enzyme's substrates is essential to understand its function, yet it remains challenging. A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic interactions. Extending this venerable concept to enzyme-catalyzed in situ adduct formation, unknown substrates were affinity-captured by an S-adenosyl-methionine (AdoMet, SAM)-dependent methyltransferase (MTase). Specifically, the electrophilic methyl sulfonium (alkyl donor) in AdoMet is replaced with a vinyl sulfonium (Michael acceptor) in S-adenosyl-vinthionine (AdoVin). Via an addition reaction, AdoVin and the nucleophilic substrate form a covalent bisubstrate-adduct tightly complexed with thiopurine MTase (2.1.1.67). As such, an unknown substrate was readily identified from crude cell lysates. Moreover, this approach is applicable to other systems, even if the enzyme is unknown.

Full text of DOI:10.1021/jacs.5b05950

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Communication
Capturing Unknown Substrates via in situ Formation of Tightly
Bound Bisubstrate Adducts: S-Adenosyl-Vinthionine as a
Functional Probe for AdoMet-Dependent Methyltransferases
Wanlu Qu, Kalli C Catcott, Kun Zhang, Shanshan Liu, Jason Jianxin Guo, Jisheng Ma, Michael Pablo,
James Glick, Yuan Xiu, Nathaniel Kenton, Xiaoyu Ma, Richard I. Duclos, and Zhaohui Sunny Zhou
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b05950 • Publication Date (Web): 22 Feb 2016
Downloaded from http://pubs.acs.org on February 22, 2016
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Capturing Unknown Substrates via in situ Formation of Tight-
ly Bound Bisubstrate Adducts: S-Adenosyl-Vinthionine as a
Functional Probe for AdoMet-Dependent Methyltransferases*
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Wanlu Qu,† Kalli C. Catcott,† Kun Zhang,‡ Shanshan Liu,† Jason J. Guo,¦ Jisheng Ma,§ Michael
Pablo,† James Glick,† Yuan Xiu,† Nathaniel Kenton,† Xiaoyu Ma,¦ Richard I. Duclos, Jr,† and
Zhaohui Sunny Zhou†
†Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeast-
ern University, 360 Huntington Ave, Boston, Massachusetts, 02115, USA.
‡School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China.
¦Center for Drug Discovery, Northeastern University, 360 Huntington Ave, Boston, Massachusetts, 02115, USA.
§School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou, 325035, China.
Supporting Information Placeholder
In practice, a major limitation for such an approach is that
substrates of multiple enzymes (“Nu” in Scheme 1) in the
ABSTRACT: Identifying an enzyme’s substrates is essential
to understand its function yet remains challenging. A fun-
same family may be labeled non-specifically, thereby the
direct association between a particular substrate-enzyme
pair cannot be readily established. This is exacerbated for
the large family of methyltransferases that catalyze over 300
different reactions with considerable overlapping of sub-
strates. For instance, more than 50 protein lysine and 9 argi-
nine methyltransferases exist in humans alone.1b-d, 1f, 1g
damental impediment is the transient interactions between
an enzyme and its substrates. In contrast, tight binding is
often observed for multisubstrate-adduct inhibitors due to
synergistic interactions. Extending this venerable concept to
enzyme-catalyzed in situ adduct formation, unknown sub-
strates were affinity-captured for an S-adenosyl-methionine
(AdoMet, SAM)-dependent methyltransferase. Specifically,
the electrophilic methyl sulfonium (alkyl donor) in AdoMet
is replaced with a vinyl sulfonium (Michael acceptor) in S-
adenosyl-vinthionine (AdoVin). Via an addition reaction,
AdoVin and the nucleophilic substrate form a covalent bi-
substrate-adduct tightly complexed with thiopurine MTase
(TPMT, EC 2.1.1.67). As such, an unknown substrate was
readily identified from crude cell lysates. Moreover, this
approach is applicable to other systems, even if the enzyme is
unknown.
Scheme 1. Methyltransferase-catalyzed transfer of a methyl
(AdoMet, natural substrate), alkyne or ketone group (sub-
strate surrogates). The traceable products can be detected
via click or oxime chemistry.
Often dubbed as Nature's machineries, enzymes play es-
sential roles in biology and diseases. Naturally, it is critical
to know what each machine breaks and builds; in other
words, the substrates and products of each enzyme. Yet
identifying the substrates of each enzyme remains challeng-
ing, notwithstanding rapid advancements in genomics, pro-
teomics and structural biology.1 One common approach is to
facilitate the detection of the products of an enzyme. To-
ward this end, substrate surrogates containing traceable tags
are widely employed, exemplified by S-adenosyl-methionine
(AdoMet or SAM)-dependent methyltransferases (MTases).1f,
Conceptually, the interaction between an enzyme and its
substrates or products is transient, i.e., being a catalyst, an
enzyme does not form a long lasting complex with either its
substrates or products, as illustrated in Scheme 2.a. As a
result, even traceable products often cannot be directly
linked to a particular methyltransferase.
2
As shown in Scheme 1, the methyl group in AdoMet has
been replaced by alkyne, ketone, and other functional groups,
which label nucleophilic substrates via the transfer of these
traceable alkyl groups; subsequently, the resulting products
can be labeled via click (azide-alkyne) or oxime (ketone-
hydroxylamine) chemistry.1
A direct, and conceptually distinct, approach is to capture
the nucleophilic substrates by the corresponding methyl-
transferase via in situ formation of bisubstrate adducts,
which should bind significantly more tightly with the
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enzyme than either substrate alone due to the synergistic
strates of TPMT all reacted with AdoVin and formed stable
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binding interactions, thereby resulting in a more persistent
complex, as illustrated in Scheme 2.b. This is the premise of
multisubstrate adduct inhibition, championed by Coward,
Pegg and others.3 This venerable concept has been ex-
plored—albeit in a few limited cases—to identify unknown
enzymes and substrates, i.e., the formation of kinase-
substrate complex via ATP-based cross-linker.4 Weinhold,
Rajski, Thompson and others developed bisubstrate-adduct
inhibitors for DNA and protein arginine methyltransferases
(PRMTs) via AdoMet analogues with 5’-aziridinyl
adenylates,1a, 2b, 5 but to our knowledge, neither the tight
binding between the adducts and MTases nor its application
on identification of unknown substrates was discussed.
adducts depicted in Scheme 3, as confirmed by HPLC-UV-Vis
and mass spectrometric analysis (see Figure S3.2.1-3.2.5).
Conversely, non-substrates toward AdoMet (e.g., substituted
phenols, e) did not react with AdoVin either. As such, the
substrate specificity toward AdoMet and AdoVin (the sub-
strate surrogate) mirrors each other, thereby satisfying a
critical requirement for such functional probes.
NO2
NH2
NO2
substrate
9
HO2C
Br
HO2C
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Br
SH
SH
SH
SH
OH
alkyl donor
AdoMet
a
b
c
e
d
non-
substrate
known
yes
known
yes
known
yes
unknown
yes
Scheme 2. (a) Transient interactions between a methyltrans-
ferase and its substrates or products during the catalytic cy-
cle; (b) persistent interaction (tight binding) between an
enzyme and the bisubstrate adduct formed in situ.
AdoVin
no
Figure 1. Same substrate specificity toward AdoVin and
AdoMet.
a)
b)
substrate
substrate
AdoVin
substrate
R
substrate
Next, tight binding of the resulting bisubstrate-adduct to
the methyltransferase was investigated. Free ligands and
enzyme-adduct complex were separated via either ultrafiltra-
tion or immobilized metal ion affinity chromatography (the
recombinant TPMT contained a hexa-histidine-tag). Under
both conditions, the AdoVin adducts were observed only in
the enzyme complex (Figure 2 and Figure S3.3.1-3.3.3), indi-
cating markedly tight binding between AdoVin adducts and
MTases, as expected from synergistic interactions between
the bisubstrate-adduct and the enzyme (Scheme 2.b). Addi-
tionally, in the presence of vast excesses of competing rea-
gents, AdoMet and AdoHcy, the adduct remains bound to
the enzyme (Figure S-3.3.5). Moreover, the sulfonium ylide
form of the adduct (Scheme S3.1.1), which closely mimics the
neutral and linear transition state of the reaction, may exist
when bound to the enzyme and thus contribute to the tight
binding.
R
AdoVin
AdoMet
AdoMet
apoenzyme
apoenzyme
adduct
formation
single
turnover
catalytic
cycle
product
R
product
bisubstrate
R
no dissociation
tight binding
AdoHcy
X
adduct
AdoHcy
Our approach is demonstrated herein with S-adenosyl-
vinthionine (AdoVin, Scheme 3), a new probe in which a
vinyl sulfonium replaces the methyl sulfonium in AdoMet.
Via an addition reaction to the vinyl sulfonium (a Michael-
type acceptor), AdoVin and the nucleophilic substrate form a
covalent tight-binding adduct.
Scheme 3. Formation of AdoVin from vinthionine and ATP
catalyzed by MAT. TPMT-catalyzed in situ formation of bi-
substrate adduct between AdoVin and thiol substrates.
thiols
R
R
SH
S
NH2
N
NH2
N
N
N
N
N
ATP
MAT
TPMT
H2N
S
S
S
N
N
O
O
H2N
H2N
CO2H
AdoVin
CO2H
AdoVin adduct
CO2H
vinthionine
OH OH
OH OH
Figure 2. HPLC chromatograms (325 nm) of (a) methyltrans-
ferase-ligand complex and (b) fractions with only free lig-
ands, showing that AdoVin-TNB adduct tightly bound with
methyltransferase, while no dissociation of the AdoVin-TNB
adduct was observed.
As illustrated in Scheme 3, AdoVin was enzymatically syn-
thesized from vinthionine and ATP catalyzed by methionine
S-adenosyl transferase (MAT, or AdoMet synthetase, EC
2.5.1.6).6 AdoVin shares similar characteristics with AdoMet
(see supplementary materials, SI).
To examine the utility of AdoVin, thiopurine methyltrans-
ferase (TPMT, EC 2.1.1.67) with a broad specificity toward
aromatic thiols, was used (Scheme 3).1e, 2a As listed in Figure
1, reduced Ellman’s reagent (TNB, a) and other known sub-
Adduct formation is both time dependent (first-order ki-
netics) and enzyme concentration dependent (Figure 3), con-
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sistent with the proposed mechanism that involves a rapid
underscores the utility of our approach in directly identifying
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initial binding of the thiol substrate and AdoVin with TPMT
and the subsequent addition reaction. The first-order rate
constant of adduct formation with AdoVin (kapp = 0.33 ± 0.12
min-1), while the kcat of transmethylation with AdoMet (13.6 ±
0.4 min-1).2a In contrast, only a single turnover formation of
the bisubstrate-adduct was observed (see Figure 3 and Figure
S3.4.2) while multiple turnovers were observed for trans-
methylation, again consistent with the markedly enhanced
binding affinity of the bisubstrate-adduct compared to the
individual components.
enzyme substrates, even unknowns.
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Figure 4. HPLC chromatograms (260 nm) of (a) ex vivo
reaction of crude cell lysate and (b) the captured AdoVin-
TNB and AdoVin-AMBA adducts from isolated TPMT com-
plex, illustrating affinity enrichment.
One general concern is whether AdoMet analogues modify
enzymes. Using mass spectrometry, no modification was
detected on either TPMT or MAT (Figure S5.2-5.3). While
vinyl sulfonium is intrinsically reactive, extensive solvation of
the highly charged sulfoniums in aqueous solution renders
low reactivity.6a, 7c, 9 For instance, no background reaction
between thiols and AdoVin was observed (see Figure S3.1.4);
and moreover, even under the ex vivo conditions where many
metabolites exist, no adducts besides the expected ones were
detected (see Figure S4.1.4).
In sum, our strategy can indeed capture and identify en-
zyme substrates, even unknowns. Conversely, if a substrate
or methylation product is known, the corresponding enzyme
can be identified as well. Applications in whole cells and
organisms can also be envisioned, as AdoVin can be synthe-
sized in vivo when vinthionine is supplemented.6c-f, 7c Addi-
tionally, previous in vivo labeling with vinthionine resulted in
modifications of a broad range of methyltransferases sub-
strates (e.g., DNA and proteins), suggesting AdoVin was ac-
cepted as a substrate by other methyltransferases.6c-f Moreo-
ver, the formation of bisubstrate adducts may also have
broad utility in facile generation of strong specific inhibitors
and structural elucidation of substrate-enzyme interactions.
Lastly, our approach can be applied toward many other en-
zymes, particularly those catalyze group transfers.
Figure 3. Changes in substrate concentration and TNB ab-
sorbance at 411 nm from the formation of adduct catalyzed by
TPMT at various concentrations.
One of our goals is to identify unknown substrates of me-
thyltransferases; in particular, no endogenous substrate for
TPMT has been reported. To this end, AdoVin was incubat-
ed with the crude cell lysate of E. coli that expressed recom-
binant human TPMT, but no adduct was detected (see Figure
S4.1.4), which was not unexpected as no aromatic thiol me-
tabolites have been reported for E. coli. As a positive control,
TNB was added to the crude cell lysate, and the correspond-
ing adduct was detected, and again, only in the enzyme
complex (see Figure 4 and Figure S4.1.1-4.1.3), indicating tight
binding under physiological conditions as well. Furthermore,
Figure 4 illustrates the drastic enrichment of the adduct even
from complex cellular mixtures. These experiments were also
carried out in HeLa cell lysates with similar results (see Fig-
ure S4.4). It is worth noting that adduct formation was ob-
served, even with competition from endogenous AdoMet
which was present in the cell lysates.
Unexpectedly, in E. coli lysates, aside from the AdoVin-
TNB adduct, another adduct (Figure 4) was detected in the
TPMT complex, but only when TNB was added to the cell
lysates, suggesting this unknown peak was derived from TNB.
Based on the UV-Vis spectrum of the unknown adduct, the
mass change (-30 Da) and fragmentation pattern of isotopic
labeled adduct (see Figure S4.1.5),7 we postulated that the
nitro-group in TNB and adduct was reduced to an amine,
which could be catalyzed by any of four nitroreductases ex-
isting in E. coli.8 This assignment was confirmed by the au-
thentic amino thiol (2-amino-5-mercaptobenzoic acid,
AMBA, d in Figure 1) and the corresponding adduct with
AdoVin (see Figure S4.2.1). It is worth noting that this amino
thiol (AMBA) had not been reported as a substrate of TPMT
but was confirmed in this work as a substrate toward AdoM-
et (see Figure S4.3.1). Altogether, this serendipitous finding
Supporting Information
Full experimental details, synthesis and characterization of
AdoVin and AdoVin adducts. This material is free of charge
via the Internet at http://pubs.acs.org.
Corresponding Author
Correspondence should be addressed to Zhaohui Sunny
Zhou, z.zhou@neu.edu.
Notes
The authors declare no competing financial interests.
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17568-17574.
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We thank NIH NIGMS (1R01GM101396 to Z.S.Z) and NIDA
(DA032020 to J.J.G.) for funding, Kevin Moulton, Dillon
Cleary, Diego Arevalo and Dharmesh Patel for helpful discus-
sions, Prof. Paul Vouros and Dr. Jared Auclair for mass spec-
trometer access, Nicholas Schmitt for help with the high-
resolution mass spectra.
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Table of Contents (TOC)
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2
3
substrate
substrate
4
5
methyltransferase
AdoVin
AdoVin
substrate
6
7
8
9
Nu
apoenzyme
adduct
formation
addition
NH2
N
single
turnover
N
N
S
N
bisubstrate
O
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H2N
no dissociation
tight binding
X
CO2H
OH OH
AdoVin
adduct
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Scheme 1. Methyltransferase-catalyzed transfer of a methyl (AdoMet, natural substrate), alkyne or ketone
group (substrate surrogates). The traceable products can be detected via click or oxime chemistry.
61x26mm (300 x 300 DPI)
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Scheme 2. (a) Transient interactions between a methyltransferase and its substrates or products during the
catalytic cycle; (b) persis-tent interaction (tight binding) between an enzyme and the bisub-strate adduct
formed in situ.
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Scheme 3. Formation of AdoVin from vinthionine and ATP cata-lyzed by MAT. TPMT-catalyzed in situ
formation of bisubstrate adduct between AdoVin and thiol substrates.
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Figure 1. Same substrate specificity toward AdoVin and AdoMet.
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Figure 2. HPLC chromatograms (325 nm) of (a) methyltransfer-ase-ligand complex and (b) fractions with
only free ligands, show-ing that AdoVin-TNB adduct tightly bound with methyltransferase, while no
dissociation of the AdoVin-TNB adduct was observed.
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Figure 3. Changes in substrate concentration and absorbance at 411 nm from the formation of adduct
catalyzed by TPMT at various concentrations.
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Figure 4. HPLC chromatograms (260 nm) of (a) ex vivo reaction of crude cell lysate and (b) the captured
AdoVin-TNB and Ado-Vin-AMBA adducts from isolated TPMT complex, illustrating affinity enrichment.
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Table of Contents (TOC)
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