Engineered SAM Synthetases for Enzymatic Generation of AdoMet Analogs with Photocaging Groups and Reversible DNA Modification in Cascade Reactions

Article abstract of DOI:10.1002/anie.202012623

Methylation and demethylation of DNA, RNA and proteins has emerged as a major regulatory mechanism. Studying the function of these modifications would benefit from tools for their site-specific inhibition and timed removal. S-Adenosyl-L-methionine (AdoMet) analogs in combination with methyltransferases (MTases) have proven useful to map or block and release MTase target sites, however their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered a SAM synthetase from Cryptosporidium hominis (PC-ChMAT) for efficient generation of AdoMet analogs with photocaging groups that are not accepted by any WT MAT reported to date. The crystal structure of PC-ChMAT at 1.87 ? revealed how the photocaged AdoMet analog is accommodated and guided engineering of a thermostable MAT from Methanocaldococcus jannaschii. PC-MATs were compatible with DNA- and RNA-MTases, enabling sequence-specific modification (“writing”) of plasmid DNA and light-triggered removal (“erasing”).

Full text of DOI:10.1002/anie.202012623

A Journal of the Gesellschaft Deutscher Chemiker
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International Edition Chemie
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Accepted Article
Title: Engineered SAM synthetases for enzymatic generation of
AdoMet analogs with photocaging groups and reversible DNA
modification in cascade reactions
Authors: Freideriki Michailidou, Nils Klöcker, Nicolas Cornelissen, Rohit
K. Singh, Aileen Peters, Anna Ovcharenko, Daniel Kümmel,
and Andrea Rentmeister
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RESEARCH ARTICLE
Engineered SAM synthetases for enzymatic generation of AdoMet
analogs with photocaging groups and reversible DNA
modification in cascade reactions
Freideriki Michailidou,†[a, b] Nils Klöcker,†[a] Nicolas V. Cornelissen,†[a] Rohit K. Singh, Aileen Peters,
[a]
[a]
[a]
[a]
[a]
Anna Ovcharenko, Daniel Kümmel, Andrea Rentmeister*
Dedication ((optional))
[a]
Dr. F. Michailidou, N. Klöcker, N. V. Cornelissen, Dr. R. K. Singh, Aileen Peters, A. Ovcharenko, Prof. Dr. D. Kümmel, Prof. Dr. A. Rentmeister
Department of Chemistry
Institute of Biochemistry
University of Münster
Corrensstr. 36, 48149 Münster, Germany
[b]
Current address: ETH Zürich, Department of Chemistry and Applied Biosciences, Laboratory of Organic Chemistry, Vladimir-Prelog-Weg 1-5/10, CH-
8093 Zürich, Switzerland
E-mail: a.rentmeister@uni-muenster.de
These authors contributed equally.
†
Supporting information for this article is given via a link at the end of the document. ((Please delete this text if not appropriate))
Abstract: Methylation and demethylation of DNA, RNA and proteins
timepoint with an orthogonal trigger would enable the in-depth
studies required to understand their function in greater detail[
.
1d]
has emerged as a major regulatory mechanism. Studying the function
of these modifications would benefit from tools for their site-specific
inhibition and timed removal. S-Adenosyl-L-methionine (AdoMet)
analogs in combination with methyltransferases (MTases) have
proven useful to map or block and release MTase target sites,
however their enzymatic generation has been limited to aliphatic
groups at the sulfur atom. We engineered a SAM synthetase from
Cryptosporidium hominis (PC-ChMAT) for efficient generation of
AdoMet analogs with photocaging groups that are not accepted by
any WT MAT reported to date. The crystal structure of PC-ChMAT at
Photocaging groups are powerful tools for probing
[
4]
biomolecular interactions and functions. They have been
successfully applied for DNA, RNA, and proteins in vitro, in cells
and in vivo[
4a, 4b]
. Their removal by light reconstitutes the native
biomolecule and can be controlled with excellent spatio-temporal
precision. The 2-nitrobenzyl (ONB) group and its derivatives are
widely used due to their stability and accessibility by chemical
[
5]
[6]
synthesis . We and others recently showed that benzylic and
photocaged (PC) AdoMet analogs[5b, 7] are converted by MTases
that are not sterically constrained and thus can be used to block
the enzymes’ target sites. The action of an MTase for the
installation of a PC group (‘writing’) can thus be combined with
light for the removal of the introduced label (‘erasing’)—an
important step towards investigations of epigenetic modifications
with spatio-temporal control (Scheme 1). However, this approach
is limited due to the degradation of AdoMet analogs in aqueous
1.87
Å
revealed how the photocaged AdoMet analog is
accommodated and guided engineering of a thermostable MAT from
Methanocaldococcus jannaschii. PC-MATs were compatible with
DNA- and RNA MTases, enabling sequence-specific modification
(“writing”) of plasmid DNA and light-triggered removal (“erasing”).
solution, their cell impermeability and the fact that their chemical
synthesis yields epimeric mixtures.[8] These issues can be
Introduction
circumvented by the enzymatic in situ generation of AdoMet
Methylation of DNA, RNA and histones is often reversible and
constitutes a regulatory mechanism with direct implications in
analogs
from methionine
analogs
using methionine
adenosyltransferase (MAT) (also termed SAM-synthetase) in vitro
[
1]
fundamental biological processes and human disease . This
epigenetic mark is introduced by methyltransferases (MTases),
which typically use S-adenosyl-L-methionine (SAM or AdoMet) as
or in cells[6a, 9]
.
To this date, neither wildtype nor engineered MATs were
reported to accept benzylic or photocaging groups,[6a, 10] although
[
2]
5
methyl donor . In DNA, m C (5-methylcytosine) leads to
inactivation of transcriptional start sites, while its oxidative
this activity would be imperative to implement this optochemical
concept in a biological setting. We sought to address this
shortcoming and develop MAT variants (PC-MATs) able to
efficiently convert methionine analogs bearing photocaging
groups (1a-c) to the respective AdoMet analogs (2a-c) (Scheme
6
6
removal recovers gene expression. Recently, m A (N -
methyladenosine) in DNA was shown to be involved in
transcriptional activation and silencing[2d, 3]. The ability to block
such methyltransferase target sites and release them at a defined
1). Such PC-MATs should enable cascade reactions with various
promiscuous MTases for the installation of photocaging groups in
1
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RESEARCH ARTICLE
MTase target molecules and their light-induced removal (Scheme
25% of 4d. The single substitution led to a 5-fold increase
compared to the WT ChMAT. The identity of 4d was confirmed by
LC-MS analysis (Figure S5). If the enzyme was left out, no
product was formed (Figure S4).
1).
(
)
Scheme 1. Concept for enzymatic in situ generation of AdoMet analogs with
photocaging groups (PC). The engineered PC-MATs enable the reversible
modification of MTase target sites, as exemplified for DNA (‘writing’) starting
from indicated methionine analogs (1) with photcaging (PC) groups and ATP in
cascade reactions. The modifications can be removed (‘erasing’) by light as an
orthogonal trigger.
Results and Discussion
For the rational re-design of the Cryptosporidium hominis MAT
(
(
ChMAT) active site, we evaluated the crystal structure of ChMAT
PDB ID: 4ODJ).[ The AdoMet binding pocket is formed at the
11]
interface of the two monomers (Figure 1A), as often observed for
MATs.[12] The methyl moiety of AdoMet is contained within a
hydrophobic pocket, which is lined by the residues I122, C125,
V126, and I330 in ChMAT (Figure 1B). Stacking interactions
between the adenine and a phenylalanine (F258) further stabilize
binding of AdoMet or ATP, respectively. These features are in line
with structures from other MATs that were analysed in
comparison (Figure S1). Furthermore, previous characterizations
of MATs revealed that a “gating loop” provides access to the
active site in a dynamic manner.[9b, 12b, 12c, 13] This gating loop is
formed by residues 118–130 in ChMAT (highlighted in green in
Figure 1A).
To expand the substrate scope of ChMAT towards benzylic
methionine analogs, we substituted the amino acids I122, V126
and I330 (that constitute the hydrophobic binding pocket) as well
as Q121 (that is part of the gating loop) by less sterically
demanding residues, generating eight ChMAT variants: I122A,
I122V, 122G, V126G, V126A, I330A, I330V and Q121A. These
variants were tested in an enzymatic cascade reaction with Ecm1,
(
(
))
Figure 1. Engineering ChMAT for activity on benzyl- (1d) and 2-nitro-benzyl-
homocysteine (1a). A) Overall structure of WT ChMAT (4ODJ). The flexible
gating loop shown in green. Magnesium ions are shown as green spheres. B)
Hydrophobic ‘pocket’ surrounding the methyl moiety of AdoMet (shown as
sticks). Residues I122, I330, V126 and C125 that form the pocket, as well as
Q121 and F258 involved in direct interactions with the adenine moiety are also
shown as sticks.
a
highly promiscuous guanine N7 methyltransferase from
Encephalitozoon cuniculi that efficiently converts benzylic AdoMet
analogs[6c, 7a, 14] (Figure 2).
As expected, the WT ChMAT enzyme showed no activity on
2
(
-nitro-benzyl-D,L-homocysteine 1a in an HPLC-based assay
0.7±0.4% conversion; Figure 2B, S3). We therefore switched to
benzyl-D,L-homocysteine 1d as surrogate substrate, and found
% conversion of 1d to the benzylated GpppA 4d according to
We anticipated that the improved activity of 1d would shift the
substrate spectrum of the variants towards substituted benzylic
moieties as previously observed in directed evolution.[15]
5
Consequently, we tested ChMAT I122A (the best variant on 1d)
for activity on a methionine analog with a photocaging group at
the sulphur atom. Indeed, ChMAT I122A showed activity on 1a,
yielding 5% conversion to 4a (Table S3). However, most of the
other variants tested (I122V, I122G, the double variant
I122V/V126A, Q121A nor V126) did not result in product
formation. To our delight, variant I330A yielded 23±3 % and the
double variant I122A/I330A even 69±14 % of 4a (Figure 2B,C).
Targeting an additional residue of the gating loop, did not lead to
further improvements, as 69±8 % of 4a were formed by the triple
variant I122A/ C125A/I330A. The formation of product 4a was
HPLC analysis (Table S3, Figure S3).
Testing the eight active site variants mentioned above, we
found that four of them – namely I122A, I122V, I122G and V126A
–
(
displayed increased activity on 1d compared to WT ChMAT
Table S3). Three variants (V126G, I330A, I330V) were active but
did not show increased activity on 1d. Q121A was not active on
d. These data suggest that positions I122 and V126 are suitable
1
sites to increase the ChMAT activity on benzylic methionine
analogs. However, combining the two beneficial mutations in
double variant I122V/V126A did not further increase activity on 1d.
Thus, ChMAT I122A showed the highest activity on 1d, yielding
2
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RESEARCH ARTICLE
confirmed by LC-MS (Figure S6). These three ChMAT variants
were also tested on methionine analogs bearing red-shifted ONB-
derivatives, namely 2-nitro-4-bromo-benzyl-homocysteine (1b)
and 2-nitro, 4,5-dimethoxy-benzylhomocysteine (1c). The
conversion to the respective products yielded up to 23±7 % 4b or
c in situ and coupled the reaction to a promiscuous DNA
methyltransferase from Thermus aquaticus (MTaqI), which
modifies the N position of adenosine, recognizing the sequence
6
5'-TCGA-3′. Subsequent light irradiation should trigger release of
the PC group, thus mimicking an epigenetic process by enzymatic
writing and light-induced erasing (Scheme 1). To this end, a short
dsDNA substrate or the plasmid pBR322, respectively, was
incubated with the respective methionine analogs 1a-c, the PC-
MAT and MTaqI, followed by enzymatic degradation to single
15±4 % 4c in cascade reactions according to HPLC analysis
(
Figure 2C,D), suggesting that additional substituents interfere
with accommodation in the active site. The respective products
were confirmed by LC-MS (Figure S7-8). The best variant
I122A/I330A was termed PC-ChMAT.
6
nucleosides and LC–MS-based analysis. In all cases, the N -
modified 2′-deoxyadenosine (5a-c) was observed, demonstrating
successful enzymatic installation of the respective PC-group. In
parallel, the identical sample was subjected to irradiation by light
(
365 nm for 5a, 405 nm for 5b-c). No modified nucleoside could
be detected under these conditions, demonstrating successful
and complete photodeprotection (Figures S9-11). In control
experiments without enzyme, the photocaged nucleosides were
not detectable either (Figures S9-11).
To understand how PC-ChMAT (ChMAT I122A/I330A) can
accommodate the ONB group of 2a and how it demonstrates a
different substrate tolerance than ChMAT I122A, we crystallized
the former enzyme variant. We obtained structures of the enzyme
in the apo form (6LTW, 1.65 Å) and with product 2a bound (6LTV)
at a resolution of 1.87 Å (Table S2). Compared to the WT, the
overall fold of the enzyme is similar, with an r.m.s.d of 1.2 Å for
apo and 0.85 Å for 2a-bound PC-MAT. In apo PC-MAT, the gating
loop is disordered and we could only model the magnesium ions
and two phosphates in the active site (Figure 3A, cyan). The
ligand-bound structure was obtained by soaking of apo crystals
with 2a and PPPi (Figure 3). Here, clear electron density for both
products was observed (Figure S11). The overall structure of the
active site is not altered by the substitutions. Interestingly, binding
of 2a induces only partial folding of gating loop residues (118-121),
which is conveyed by the hydrogen bond interactions of Q118 with
the carboxyl group and a water-mediated backbone amide
interaction with the nitro group of the product (Figure S12).
Because parts of the gating loop engage in crystal contacts,
disorder in the gating loop might be fostered by crystal packing.
As in WT-ChMAT, F258 interacts with the adenine base via
stacking interactions. The ONB group of PC-AdoMet 2a also
engages in an intra-molecular stacking interaction with the
adenine ring (Figure 3B). This conformation provides a stabilizing
interaction and, at the same time, minimizes the potential for steric
clashes with amino acid side chains in proximity. Indeed, the
substituted amino acid A330 shows a distance of 4-4.1 Å from the
ONB benzyl ring (Figure 3B). The superposition of the structures
for the variant complexed with 2a and the WT complexed with
AdoMet shows that the substitution of residue I330 to alanine
indeed decreased the steric hindrance in the active site (Figure
3C). The nitro group of ONB would still clash with the side chain
)
of A122 if the gating loop was closed. This suggests that its partial
unfolding might support turnover of unnatural AdoMet analogs,
consistent with the larger effect of the I330A substitution
compared to I122A in production of 4a (Table S3). The inverse
effect was observed for production of 4d (Table S4), which does
not contain the nitro group. Here, we envision an alternative
binding mode, where the gating loop is closed and substitution of
I122 to alanine allows accommodation of benzyl in the active site,
but I330 is required to form a tight hydrophobic pocket. These
data reveal the molecular basis for the ability of PC-ChMAT to
accommodate PC-AdoMets.
Figure 2. A) Enzymatic cascade reaction with ChMAT variants, Ecm1, and
methionine analogs 1a-d leading to formation of 4a-d respectively. B) HPLC
runs for conversion of substrate 1a in a cascade reaction with WT-ChMAT or
variant I122A/I330A (PC-ChMAT). C-D) Conversions obtained with PC-ChMAT
(C) and triple variant ChMAT I122A/C125A/I330A (D) for indicated products.
Data and error bars show average values and std. dev. of at least three
independent experiments.
To test whether PC-ChMAT was also compatible with other
MTases, we sought to assess its utility in an enzymatic cascade
reaction with a DNA MTase. We thus generated PC-AdoMets 2a-
3
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
data show that our structural model was used successfully to
design a thermostable archaeal PC-MAT.
We anticipated that the thermostable PC-MAT should be
ideal for cascade reactions with MTases with optimal
temperatures at elevated temperatures, such as MTaqI
mentioned above. We used plasmid DNA containing four MTaqI
recognition sites and carried out the enzymatic cascade with PC-
MjMAT/MTaqI starting from methionine or 1a and ATP. If all sites
are methylated or modified with the photocaging group, the
plasmid is protected from restriction by the corresponding
restriction enzyme R. TaqI, which recognizes the same sequence
motif (Figure 4C) and only linearization by Nde I takes place.
Indeed, modification starting from methionine or 1a led to
protection of the plasmid (Figure 4D), indicating that PC-MjMAT
efficiently generates 2a that is readily used by MTaqI. Irradiation
of the modified plasmid did not have an effect in the case of
methylation, but led to restriction by R.TaqI in the case of
modification with the ONB group (Figure 4D). Taken together
these data show for the first time sequence-specific modification
of DNA (“writing”) in a MAT/MTase cascade with a photo-caging
group. Thus, sequence-specific enzymatic writing becomes
compatible with light-triggered “erasing” starting from methionine
analogs.
(
(
Figure 3. A) Superimposed structures of WT-ChMAT (4ODJ, shown in yellow)
with product bound PC-ChMAT (6LTV, shown in grey) and apo PC-ChMAT
(
(
6LTW, shown in cyan). B) Ligand binding by PC-ChMAT. Residues A330, F258
grey), PC-AdoMet 2a (grey) and triphosphate (orange) are shown as sticks,
2+
while Mg is shown as ball (green). Structure of apo PC-ChMAT (cyan) is
shown in comparison. C) Superimposed active sites of PC-ChMAT with 2a
(
grey) and WT-ChMAT with AdoMet (yellow). Residues I122, I330, F258,
2+
ligands and triphosphate (orange) are shown as sticks, while Mg ions are
shown as spheres (green). PC-AdoMet 2a binding to WT-ChMAT would cause
steric clashes with I122 and I330. D) Modeled active sites of PC-MjMAT
Conclusion
(
bearing equivalent mutations to PC-ChMAT) with 2a (grey). Residues A351,
2+
In conclusion, structure-guided protein engineering was
successful to obtain PC-MATs, the first MAT variants reported to
accept methionine analogs with photocaging groups. We showed
that the PC-methionine analogs can be efficiently utilized by
different MAT enzymes, significantly expanding the substrate
scope of MATs. We can now photocage plasmid DNA in a
sequence-specific manner starting from the metabolic AdoMet
precursors using enzymatic MAT/MTase cascades and
subsequently release it by irradiation. A detailed crystallographic
analysis of the biocatalyst provides insights into the structural
determinants for the expanded substrate spectrum of the variant
I122A/I330A. The latter was used to model PC-MjMAT and
represents a parent for further enzyme optimization. Mutations
were required to generate an enlarged hydrophobic pocket that
accommodates the ONB group, which is coordinated by stacking
interactions with the adenine base and hydrogen bonds of the
nitro group with the gating loop backbone. With further
engineering, the enzymatic cascade accepting photocaging
groups bears potential for future cellular applications by blocking
Y273, ligands and triphosphate (orange) are shown as sticks, while Mg ions
are shown as spheres (green).
For application in different enzymatic cascades, we wanted to find
out whether MATs with different temperature profiles can be
engineered based on the identified substitutions. MATs from
Archaea diverge considerably from bacterial/eukaryotic MATs[
and we turned our attention to the thermostable archaeal MAT
from Methanocaldococcus jannaschii. This MAT has previously
been reported to be promiscuous,[17] however, no activity on
methionine analogs with benzylic groups had been observed and
no crystal structure of MjMAT is available.[18] Sequence alignment
16]
suggests
that
the
substitutions
L147A/I351A
and
L147A/V150A/I351A of MjMAT would lead to variants with similar
substrate specificity as PC-ChMAT (Figure S17). Using a
homology model of MjMAT based on the crystal structure of
Sulfolobus solfataricus MAT (PDB ID: 4K0B),[
sequence identity of 51% and are highly conserved in the active
site (Figure S17), and our structure of PC-ChMAT (PDB ID: 6LTV), and releasing MTase target sites with light, which is an orthogonal
10b]
which share a
we generated a model for MjMAT L147A/I351A (PC-MjMAT)
Figure S18, 3D). Structural analysis supports that this variant can
accept 1a-d as substrates, and we thus generated the respective
MjMAT variants (Figure S16).
trigger and provides exquisite spatio-temporal control. This work
is an important step for the implementation of in situ generated
AdoMet analogs in epigenetic studies.
(
To assess their activity on 1a-c, we made use of the
previously described degradation of AdoMet analogs to
methylthioadenosine (MTA) analogs under certain conditions,[
including elevated temperature (Figure 4A). As expected, the
thermostable WT-MjMAT and PC-MjMAT converted methionine
and ATP at 65 °C, leading to MTA formation (Figure S21A/B). For
the methionine analogs 1a-c, PC-MjMAT and the triple variant
were active, generating the respective MTA analogs (5a-c),
whereas WT-MjMAT was not (Figures 4B and S21C-J). These
19]
((.
4
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RESEARCH ARTICLE
and NMR facilities of the organic chemistry department are
gratefully acknowledged for analytical services. The plasmid for
ChMAT herein was provided by the Seattle Structural Genomics
Center for Infectious Disease (www.SSGCID.org) which is
supported Federal Contract No. HHSN272201700059C from the
National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Department of Health and Human Services.
The MjMAT plasmid herein was kindly provided by Prof. Jennifer
Andexer (University of Freiburg). The synchrotron MX data was
collected at beamline P13 operated by EMBL Hamburg at the
PETRA III storage ring (DESY, Hamburg, Germany). We would
like to thank Saravanan Panneerselvam for the assistance in
using the beamline.
Keywords: MAT • SAM • protein engineering • bioorthogonal •
photocaging
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