Engineering Orthogonal Methyltransferases to Create Alternative Bioalkylation Pathways

Article abstract of DOI:10.1002/anie.202004963

S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) catalyse the methylation of a vast array of small metabolites and biomacromolecules. Recently, rare carboxymethylation pathways have been discovered, including carboxymethyltransferase enzymes that utilise a carboxy-SAM (cxSAM) cofactor generated from SAM by a cxSAM synthase (CmoA). We show how MT enzymes can utilise cxSAM to catalyse carboxymethylation of tetrahydroisoquinoline (THIQ) and catechol substrates. Site-directed mutagenesis was used to create orthogonal MTs possessing improved catalytic activity and selectivity for cxSAM, with subsequent coupling to CmoA resulting in more efficient and selective carboxymethylation. An enzymatic approach was also developed to generate a previously undescribed co-factor, carboxy-S-adenosyl-l-ethionine (cxSAE), thereby enabling the stereoselective transfer of a chiral 1-carboxyethyl group to the substrate.

Full text of DOI:10.1002/anie.202004963

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Engineering orthogonal methyltransferases to create alternative
bioalkylation pathways.
Authors: Abigail J Herbert, Sarah A Shepherd, Victoria A Cronin,
Matthew R Bennett, Rehana Sung, and Jason Micklefield
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202004963
Link to VoR: https://doi.org/10.1002/anie.202004963

10.1002/anie.202004963
Angewandte Chemie International Edition
RESEARCH ARTICLE
Engineering orthogonal methyltransferases to create alternative
bioalkylation pathways.
Abigail J. Herbert, Dr. Sarah A. Shepherd, Dr. Victoria A. Cronin, Dr. Matthew R. Bennett, Rehana
Sung and Prof. Jason Micklefield *
Department of Chemistry and Manchester Institute of Biotechnology
The University of Manchester
131 Princess Street, Manchester M1 7DN, UK
Email: jason.micklefield@manchester.ac.uk
Supporting information for this article is given via a link at the end of the document.
Abstract: S-adenosyl-L-methionine (SAM)-dependent methyltransfer
-ases (MT) are an important class of enzymes, catalysing
methylation of
a
vast array of small metabolites and
biomacromolecules. Recently, rare carboxymethylation pathways
have been discovered, including carboxymethyltransferase enzymes
that utilise a carboxy-SAM (cxSAM) cofactor, generated from SAM
by a cxSAM synthase (CmoA). In this study we show how MT
enzymes can utilise cxSAM, catalysing carboxymethylation of
tetrahydroisoquinoline (THIQ) and catechol substrates. Site-directed
mutagenesis was used to create orthogonal MT possessing
improved catalytic activity and selectivity for cxSAM, with
subsequent coupling to CmoA resulting in more efficient and
selective carboxymethylation. An enzymatic approach was also
developed to generate a previously undescribed co-factor, carboxy-
S-adenosyl-L-ethionine (cxSAE), enabling the stereoselective
transfer of a chiral 1-carboxyethyl group to the substrate. These
findings provide a platform by which common methylation pathways
can be engineered to deliver carboxymethylated products, providing
new properties and functionality.
Introduction
Methylation is one of the simplest and most widely used
reactions in nature. The majority of methylation reactions
occurring within cells are catalysed by S-adenosyl-L-methionine
(SAM) dependent methyltransferase (MT) enzymes that transfer
a methyl group from SAM to nucleophilic centres in a wide array
of substrates, including small metabolites, proteins, nucleic acids
and other biological molecules.1–5 Typically, MTs transfer methyl
groups with exquisite chemo- and regio-selectivity and often
have a profound effect on the properties of biological molecules,
making them a highly diverse and important class of enzymes.
For example, methylation of proteins and DNA is a key
mechanism in the epigenetic control of gene expression.1,2 MT
Figure 1. a) Alkyl diversification of natural products. Rapamycin and
rebeccamycin derivatives generated using MT and SAM analogues.11,12 b)
CmoA catalysed formation of cxSAM. In the native pathway, cxSAM functions
as a co-factor for the carboxymethyltransferase CmoB in the modification of a
tRNA substrate. In this work (grey), we show how CmoA can be combined
with an engineered methyltranferase (MT*), opening up new pathways to
carboxymethylated products.
catalysed methylation is also
a common theme in the
biosynthesis of therapeutically important secondary metabolites,
including antibiotics, where methyl substituents can affect the
properties and bioactivity of the natural products (NP).3,5
Several SAM-dependent MTs have been shown to accept
synthetic SAM analogues, with alternative S-alkyl, -allyl or -
propargyl substituents, facilitating non-native alkylation
reactions.5-16 The ability to transfer functional groups
regioselectively using MTs and SAM derivatives has been
exploited for the site-selective labelling of proteins and DNA,5-10
as well as in the structural diversification of clinically important
NPs, including the anticancer agent rebeccamycin and
rapamycin immunosuppressive agents (Figure 1a).11,13 In most
cases, the SAM analogues are prepared synthetically through
the alkylation of S-adenosyl-L-homocysteine (SAH); this process
however gives rise to a mixture of sulphonium-epimers, with the
natural (S)-epimer functioning as a cofactor and the (R)-epimer
potentially inhibiting activity. Some SAM analogues can be
prepared from synthetic methionine derivatives and ATP using a
methionine adenosyltransferase (MAT) enzyme to produce the
1
This article is protected by copyright. All rights reserved.

10.1002/anie.202004963
Angewandte Chemie International Edition
RESEARCH ARTICLE
biologically relevant sulphonium epimer as the sole product.11,13
MAT has also been used to produce related seleno-SAM
analogues.14,15 Notwithstanding this, SAM analogues are
expensive to produce, unstable and cannot penetrate the cell
membrane, largely limiting their utility to small scale in vitro
applications.16-20
New enzymatic approaches to produce SAM derivatives
from metabolically available, rather than synthetic, precursors
would be highly desirable. Such approaches, combined with
engineered orthogonal MTs that are selective for enzymatically
derived SAM analogues, could open up new bioalkylation
pathways in vitro and in vivo. To this end, we were particularly
interested in the discovery of a naturally occurring SAM
derivative, carboxy-S-adenosyl-L-methionine (cxSAM), which is
generated from SAM and prephenate by the cxSAM synthase
(CmoA) enzyme (Figure 1b).21,22 CmoA is found in bacterial
species and functions in tandem with
a carboxymethyl
transferase (CmoB), which transfers the carboxymethyl group
from cxSAM to a tRNA substrate.23 Whilst another pathway was
recently
discovered
which
utilises
cxSAM
in
the
carboxymethylation of a peptide substrate,24 such bioalkylation
pathways are extremely rare in nature. However, if CmoA could
be coupled with orthogonal variants of the more common MTs,
engineered to have higher selectivity for cxSAM, then a large
array of diverse carboxymethylated products could be generated
with new functionality and bioactivity. In this paper, we
demonstrate how CmoA can be utilised in tandem with MT
enzymes to generate carboxymethylated, rather than methylated,
products. Furthermore, we demonstrate how active site
mutagenesis can lead to orthogonal MTs with improved
selectivity for cxSAM over SAM, opening the way to engineering
new carboxymethylation pathways in vitro and in vivo. Finally,
we show how CmoA can also be used to generate a novel
carboxy-S-adenosyl-L-ethionine (cxSAE) cofactor, facilitating the
stereoselective MT catalysed transfer of a chiral 1-carboxyethyl
group.
Figure 2. a) Catechol carboxymethylation assays with synthetic cxSAM,
catechols 2 or 3 (0.25 mM), COMT (100 μM), MgCl2 (6 mM) and DTT (1 mM),
incubated for 20 h. b) THIQ carboxymethylation assays. CNMT was pre-
incubated with THIQ 4 or 5 for 1 h to remove co-purified SAM and then
transferred to fresh buffer. Assays were then conducted with synthetic cxSAM
(1.6 mM), substrate 4-7 (0.25 mM) and CNMT (100 μM) and incubated for 17
h. Assays were done in triplicate and standard errors calculated.
Results and Discussion
COMT Y200L providing 76% meta- and 5% para-carboxymethyl
products 3a and 3b, respectively (Figure 2a). For both catechol
substrates 2 and 3, small amounts of the corresponding
methylated products were observed (e.g. 2 with WT and Y200L
COMT gave 21 ± 1% and 16 ± 1% methylated products,
respectively), which is attributed to co-purification of SAM with
COMT and background decarboxylation of cxSAM to SAM which
is apparent during the incubation period.
Activity of methyltransferases (COMT
& CNMT) with
synthetic cxSAM. To assess the possibility of using cxSAM as
an alternative cofactor to SAM, we chose to explore reactions
catalysed by catechol-O-methyltransferase (COMT) from Rattus
norvegicus and coclaurine-N-methyltransferase (CNMT) from
Coptis japonica. COMT was previously shown to accept a range
of catechol substrates as well as synthetic SAM analogues.25
In our previous studies, we also showed that CNMT accepts
a variety of tetrahydroisoquinoline (THIQ) substrates as well as
SAM analogues.26 To further explore its cofactor selectivity, the
WT CNMT was incubated with synthetic cxSAM and various
THIQ substrates (4-7) (Figure 2b & Figure S2). THIQs 4 and 5
showed significant carboxymethylation (82 & 83% respectively),
whilst THIQs 6 and 7 gave only low conversions with cxSAM (8-
12%). Using the crystal structure of CNMT (PDB 6GKV),26 we
infer that the close proximity of the carboxymethyl group of
cxSAM and the C1-substituent of the THIQs (6 & 7) is likely to
cause steric hindrance leading to decreased activity.
Accordingly, WT
COMT
was
incubated
with
3,4-
dihydroxybenzaldehyde 2 and synthetic cxSAM, prepared by
alkylation of SAH with bromoacetic acid,21 resulting in a mixture
of meta- and para-carboxymethylated regioisomers 2a (40%)
and 2b (29%), respectively (Figure 2a & Figure S1). These
results are consistent with the relaxed regioselectivity observed
for methylation reactions catalysed by COMT.25 Previously we
showed that a COMT Y200L mutant has significantly improved
regioselectivity, affording predominately meta-methylation of
various substituted catechol derivatives.25 In light of this,
catechol 2 and cxSAM were similarly incubated with COMT
Y200L, affording significantly improved regioselectivity for meta-
over para-carboxymethylation (2a 64% vs 2b 3%). Similar
results were obtained for 4-nitrocatechol 3 and cxSAM, with
Site-directed mutagenesis of CNMT. Having established that
CNMT can utilise cxSAM as a cofactor, we sought to develop
CNMT mutants with increased activity and selectivity for cxSAM,
as SAM will ultimately be present in significant levels within
2
This article is protected by copyright. All rights reserved.

10.1002/anie.202004963
Angewandte Chemie International Edition
RESEARCH ARTICLE
Total Conversion
Para
Meta
WT
Y81R
Y81K
a
b
WT
Y81R
Y81K
c
COMT, SAM & 2
Y200
100
90
80
70
60
50
40
30
20
10
0
CmoB
100
90
80
70
60
50
40
30
20
10
0
R315
cxSAM
CNMT, SAM & 4
150 200
K91
0
50
100
250
WT
Y200L
M40A
Para
M40A
Y200L
Time (min)
WT
WT
Y81R
Y81R
Y81K
Y81K
Total Conversion
Meta
CNMT
E207
COMT, cxSAM & 2
70
60
50
40
30
20
10
0
45
40
35
30
25
20
15
10
5
Y81
E204
CNMT, cxSAM & 4
cxSAM
0
0
50
100
150
200
250
WT
Y200L
M40A
M40A
Y200L
Time (min)
Figure 3. a) Crystal structure of CmoB and a model of cxSAM binding into the CNMT active site. CmoB active site (top) highlights three residues thought to
interact with the carboxymethyl group (PDB 4QNU). WT CNMT (bottom) with cxSAM positioned in the active site (based on PDB 6GKV which has SAH bound),
highlighting three residues predicted to be in proximity to the carboxymethyl group. b) CNMT time courses with THIQ 4 (0.25 mM) and SAM or cxSAM were
conducted using either 1 mM SAM (top/blue) or 1.6 mM cxSAM (bottom/red) due to commercial SAM comprising of only the active (S)-enantiomer at 80% purity
and cxSAM synthesised as a mixture of enantiomers. c) COMT activity assays with catechol 2 (0.2 mM) and SAM or cxSAM were conducted for 5 h at 37 ˚C to
compare % methylation and carboxymethylation, using either 1 mM SAM (top/blue) or 1.6 mM cxSAM (bottom/red). Assays were done in triplicate and standard
errors calculated.
tandem CmoA-CNMT cascade reactions. CmoB, which naturally
functions in tandem with CmoA, exhibits ca. 500 fold higher
affinity for cxSAM over SAM, which presumably ensures that no
significant competing methylation of its tRNA substrate occurs in
vivo.21,23 In light of this, we used the reported X-ray crystal
structure of CmoB (PDB 4QNU) to guide mutagenesis of
CNMT.26 Due to the low sequence similarity between CNMT and
CmoB (17.5% identity matrix, CLUSTAL 2.1), only those
residues in close proximity to the cofactor were considered.
Within the CmoB active site Lys91, Tyr200 and Arg315 residues
were identified, which form interactions with the S-
carboxymethyl group of cxSAM, likely contributing to the high
selectivity of CmoB (Figure 3a).23 A model of CNMT in complex
with cxSAM, possessing a natural (S)-configured sulphonium
centre, indicates that Glu204, Glu207 and Tyr81 residues would
be in closest proximity to the S-carboxymethyl group. These
residues were each subjected to site-directed mutagenesis,
introducing either smaller residues which could provide more
space to accommodate the larger carboxymethyl group, or basic
residues which may form favourable electrostatic (salt-bridge)
interactions with the carboxylic acid moiety.
it may be important for substrate and/or cofactor binding via
electrostatic interactions.26 In contrast, all of the Y81 mutants
showed comparable or higher activity with cxSAM and
significantly lower activity with SAM, compared with the WT, with
the Y81R mutant showing greatest cxSAM selectivity (Figure S3
& 4). At this stage we have not pursued determination of
accurate kinetic parameters as the synthetic cxSAM produced is
a
mixture of diastereoisomers, separation of which is
problematic due to the instability of the co-factor. In addition, as
stated above CNMT co-purifies with SAM, which would further
complicate determination of accurate kinetic parameters. We
instead looked to demonstrate enzyme selectivity through
separate time course assays for WT and Y81R/K CNMT with
SAM or cxSAM and substrate 4 (Figure 3b). This showed that
methylation of 4 with SAM by the WT CNMT is complete in 30
min, whilst both Y81R/K mutants afford <25% methylation over
the same time period. In contrast, carboxymethylation of 4 with
cxSAM is 23% and 28% higher than the WT for Y81K and Y81R,
respectively, across the 0-4 hour time period. The observation
that Y81K affords lower levels of carboxymethylated product
(4a), compared with the WT, in 17 h assays described above
(Figure 2a) may be due to the instability of the Y81K mutant
Eleven CNMT mutants in total (E204A/S/K, E207G/S/K/R &
Y81F/H/K/R,) were assessed for activity with THIQ 4 and cxSAM. over the longer time course.
In order to reduce competing methylation occurring due to co-
purification of SAM with CNMT, the enzymes were pre-
incubated for an hour with THIQ 5 and washed with reaction
buffer prior to assay with cxSAM and 4. All E204 & E207
mutants tested showed complete loss of activity with cxSAM.
The E204 residue is situated close to the sulphonium centre of
the cofactor and ammonium ion of the substrate, which suggests
In addition, competition assays varying ratios of SAM:cxSAM,
with THIQ substrate 4, also reveal that mutants Y81R/K have
improved selectivity for cxSAM (Figure S5). We hypothesise
that the basic side chains of R/K81 mutants may be suitably
positioned to form polar interactions with the carboxyl group of
cxSAM similar to the interactions observed in CmoB.19,21 Taken
together, the data presented here provides a proof-of-concept
3
This article is protected by copyright. All rights reserved.

10.1002/anie.202004963
Angewandte Chemie International Edition
RESEARCH ARTICLE
that the selectivity of MTs for cxSAM can be significantly
improved by rational targeted mutagenesis.
cxSAM could be generated if CM was omitted from the reaction
(Figure S8 & 9). Chorismate is well known to undergo a
spontaneous Claisen rearrangement to prephenate, and
although CM accelerates this reaction a million-fold, the rate for
the non-enzymatic rearrangement is sufficient for efficient CmoA
catalysed SAM carboxylation. Following optimisation, a tandem
coupled assay was developed that utilises chorismate and
CmoA to give cxSAM (ca. 80%), with some residual SAM (ca.
20%) remaining, followed by addition of CNMT and the substrate
4. Through this methodology, CmoA and the more cxSAM
selective CNMT Y81R mutant afforded 70 ± 1% conversion of
THIQ 4 to carboxymethylated product 4a, with only 16 ± 1%
methylated THIQ 4b produced (Figure 4b, Figure S10 & 11).
Whilst not completely selective, the final 4.5:1 ratio of
carboxymethylation to methylation observed with CmoA-CNMT
(Y81R), compares favourably with the coupled assays with
CmoA-WT CNMT which give a 1:1 mixture of carboxymethylated
to methylated products. As both SAM and prephenate are
present in bacterial cells, we envisage that the combination of
CmoA and an engineered MT, with higher selectivity for cxSAM
over SAM, may have potential to generate carboxymethylated
products in vivo as well as in vitro.
Site directed mutagenesis of COMT. A similar approach was
adopted to engineer COMT variants with higher selectivity for
cxSAM. Within the active site of COMT we observed a
methionine residue (M40) in close proximity (2.7 Å) to the methyl
group of SAM (Figure S6). Accordingly, six point mutants were
produced (M40K/R/H/A/S/C) in order to alter the electrostatic
and steric interactions of this residue with cxSAM. Of these
mutants, M40A showed the most significant increase in activity
with cxSAM (Figure 3c & Figure S7). We propose that this is
due to the increased space surrounding the carboxymethyl
group of cxSAM within the active site, leading to reduced steric
hindrance. Whilst M40A has increased activity with cxSAM, the
regioselectivity of carboxymethylation was poor. As the COMT
Y200L mutant is known to achieve high regioisomeric excess
(re), >90% meta,25 we produced an M40A/Y200L double mutant.
Assays conducted over five hours showed that the double
mutant has increased regioselectivity with cxSAM, affording
meta-carboxymethylation with high re (94%) (Figure 3c).
Moreover, M40A/Y200L also showed a 21% decrease in
methylation yields when assayed with SAM, compared to the
WT. Although both the M40A and M40A/Y200L mutants
exhibited improved selectivity for cxSAM vs. SAM, further
mutagenesis may be necessary to deliver COMT variants with
higher selectivity for more efficient in vitro or in vivo
carboxymethylation reactions.
Carboxy-S-adenosyl-L-ethionine
(cxSAE)
and
CNMT
catalysed carboxyethylation. In order to further expand the
synthetic utility of CmoA-MT cascade reactions, we envisaged
generating
a new cofactor carboxy-S-adenosyl-L-ethionine
(cxSAE), which may enable MT-mediated transfer of a chiral 1-
carboxyethyl group (Figure 5a). To test this, synthetic cxSAE,
generated from alkylation of SAH with (±)-2-bromopropionic acid
(BPA), was incubated with CNMT WT, Y81K and Y81R mutant
enzymes (Figure S12). The WT, Y81K and Y81R enzymes
showed significant activity with synthetic cxSAE, giving 25, 13
and 32% of 1-carboxyethyl THIQ 4c respectively. To determine
the configuration of the product 4c, the substrate THIQ 4 was
also separately alkylated with (S)- and (R)-BPA. However, chiral
HPLC analysis revealed that the alkylation of 4 proceeds with
significant racemisation. This is likely due to enolisation of BPA
at the elevated temperature of the reaction (>40°C).29 In addition
BPA can form an -lactone which could lead to the alkylation of
4 proceeding with overall retention of configuration, whilst direct
alkylation with BPA proceeds with inversion of stereochemistry.
In light of this, 4 was alkylated with (R)- and (S)-BPA methyl
esters and then hydrolysed to afford 1-carboxyethyl THIQ
standards, (S)-4c and (R)-4c, in high enantiomeric excesses
(ee) (95% ee in each case). Using these standards it was
apparent, from chiral HPLC, that CNMT catalysed
carboxyethylation of THIQ 4 with synthetic cxSAE, generated
from racemic BPA, is completely stereoselective giving only (R)-
4c. To further probe the stereochemistry of this process,
synthesis of cxSAE was also attempted using homochiral BPA
methyl esters, however alkylation reactions in water or mixed
aqueous-organic solvents proved problematic. Therefore, SAH
was separately alkylated with (R)- and (S)-BPA at low
temperature to minimise racemisation and the resulting cxSAEs
were similarly incubated with WT, Y81K and Y81R CNMT and
THIQ substrate 4. Whilst the yields of 1-carboxyethyl THIQ were
similar for (±)- and (S)-BPA derived cxSAE, the yields with (R)-
BPA derived cxSAE were significantly reduced. Surprisingly,
however, the (R)-configured carboxyethyl THIQ product (R)-4c
was formed in all cases (Figure 5b).
CmoA-MT coupled assay. We next sought to couple CmoA
with CNMT to generate cxSAM enzymatically for
carboxymethylation of the THIQ substrate 4 in a cascade
reaction (Figure 4a). Initially, we envisaged using chorismate
mutase (CM) to generate prephenate from chorismate, which
can be isolated in high yield from an E. coli KA12 CM-deletion
strain.27,28 However, after optimising conditions for production of
cxSAM from SAM and chorismate with CM and CmoA, it was
apparent that equivalent and in some cases higher yields of
[I]
4b
4
4a
[II]
[III]
[IV]
9
10
11
12
Time/min
Figure 4. a) Enzymatic cascade reaction for in vitro THIQ carboxymethylation.
CM† can be omitted from the reaction as the rearrangement of chorismate to
prephenate can proceed spontaneously. b) HPLC traces of cascade reactions.
[I] mixed standards; [II] assay of WT CNMT and CmoA without optimisation;
[III] assay of CNMT Y81R and CmoA without optimisation; [IV] assay of CNMT
Y81R and CmoA following optimisation as shown in Figure S10 E. HPLC
peaks colour coded: grey is starting material 4; red is carboxymethylation
product 4a; blue is methylation side product 4b.
4
This article is protected by copyright. All rights reserved.

10.1002/anie.202004963
Angewandte Chemie International Edition
RESEARCH ARTICLE
(S)-4c
(R)-4c
synthetic
standard
( )-BPA
cxSAE
Conversion of 4 to 4c (%)
(R)-BPA
cxSAE
(±)-BPA
(S)-BPA
(R)-BPA
WT
Y81K
13 ± 1
20 ± 1
8 ± 1
Y81R
32 ± 2
34 ± 1
12 ± 1
cxSAE
25 ± 1
25 ± 1
10 ± 1
(S)-BPA
cxSAE
5
6
7
Time/min
Figure 5. a) Routes to in vitro carboxyethylation. Synthesis of cxSAE from SAH and BPA, or enzymatic production of cxSAE using hMAT and CmoA, followed by
subsequent CNMT catalysed carboxyethylation of THIQ 4. The table insert shows C18 HPLC conversions of THIQ 4 to (R)-4c when assayed with WT, Y81R and
Y81K CNMT enzymes and synthetic cxSAE generated from (±), (S) or (R)-BPA. Assays were done in triplicate and standard errors calculated. b) Independent
chiral HPLC assay analysis of carboxyethylated heliamine products from CNMT Y81K and (±), (S) or (R)-BPA derived synthetic cxSAE.
Four diastereoisomers of cxSAE can be produced upon
alkylation of SAH with BPA. However, the separation of these
diastereoisomers and subsequent determination of their
absolute configurations is very difficult, particularly given their
instability. Consequently it was not possible to reach any
conclusions about the stereochemical course of the process
using synthetic cxSAE. To avoid the complication of using
diastereoisomeric mixtures of cxSAE we attempted to generate
cxSAE enzymatically, which should afford only one stereoisomer
of cxSAE. Firstly, a human methionine adenosyltransferase
(hMAT2A) mutant (I322V), known to show promiscuity with
methionine derivatives,11,17 was used to form S-adenosyl-L-
ethionine (SAE) from ATP and ethionine (Figure 5a). CmoA was
then added along with prephenate to form cxSAE in 13% yield.
The enzymatic cxSAE was then incubated with CNMT and THIQ
4, leading to production of carboxyethyl THIQ 4c. As with the
synthetic cxSAE, only the (R)-4c was evident from chiral HPLC-
MS (Figure S13). Based on the crystal structure of CmoA in
complex with cxSAM (PDB 4GEK) and computational
predictions of likely positions of precursors SAM and prephenate
in the CmoA active site,21 we predict that cxSAE would possess
1-(S)-carboxyethyl group and an (S)-configured sulphonium
centre (Figure S14). In light of this, we conclude that the CNMT
carboxyethylation of THIQ 4, with cxSAE, proceeds with
inversion of configuration, which is also consistent with the
native methylation reactions of MT which have been shown to
proceed with inversion using SAM possessing the chiral methyl
group.28 Taken together, these results suggest that CxSAE
possessing a 1-(R)-carboxyethyl group, generated synthetically
from BPA, is not turned over by CNMT.
knowledge, there are no other reports describing how CmoA can
be combined with MTs to create new pathways to
carboxymethylated rather than methylated products. Previous
approaches to produce SAM analogues, as co-factors for MT,
have relied on multi-step chemical or enzymatic synthesis for in
vitro applications which are limited by the costs of precursor and
the instability of SAM analogues. Our approach opens up the
possibility of combining CmoA and engineered MTs to create
new structural diversity, in vitro and in vivo, from entirely natural
precursors, obviating the need for multistep chemical or
enzymatic synthesis and purification steps.
MTs are amongst the most common enzymes in nature,
methylating a vast array of substrates, from small metabolites to
biomacromolecules. Addition of
a
charged and polar
carboxymethyl group, rather than a small hydrophobic methyl
substituent, could therefore lead to many new products with
significantly altered physiochemical properties, biological
activities and potentially new functions. Moreover, the additional
carboxyl group provides
a
handle for further selective
derivatisation using conventional amide coupling chemistry or
powerful metallaphotoredox reactions,31–34 that can be
performed under mild aqueous (biocompatible) conditions.35
Such downstream chemistry could be used to fine tune the
properties of bioactive molecules and can provide further
opportunities for regioselective bioconjugation and labelling. 5-10
Finally, we have demonstrated that CmoA can be used to
generate a new co-factor, cxSAE, which was accepted by CNMT
facilitating stereoselective transfer of a chiral 1-carboxyethyl
group to a THIQ substrate. Several C-methyltransferases,
including GlmT and MppJ,36-38 have been described that transfer
a methyl group to a prochiral substrate creating a new
stereogenic centre. However, we are unaware of any other
examples where a MT has been shown to catalyse the
stereoselective transfer of a chiral functional group from the co-
factor to the substrate.
Conclusion
In summary, we have shown that two typical members of the
ubiquitous Class I methyltransferase, COMT and CNMT, can
utilise cxSAM as an alternative cofactor to generate
carboxymethylated products. Structure guided mutagenesis was
used to engineer COMT and CNMT mutants with improved
selectivity for cxSAM. This enabled a tandem enzyme reaction
to be developed with the cxSAM synthase (CmoA) and CNMT
delivering carboxymethyl-THIQ in good yields. To the best of our
Acknowledgements
We acknowledge BBSRC (grant BB/K00199X/1) and GSK for
financial support and a PhD studentship to VC. We thank Peter
Sutton and Joe Adams (GSK) for helpful discussions.
5
This article is protected by copyright. All rights reserved.

10.1002/anie.202004963
Angewandte Chemie International Edition
RESEARCH ARTICLE
Keywords: Methyltransferase; carboxymethylation;
bioalkylation; cofactors; biotransformations.
[1] F. Lyko, Nat. Rev. Genet. 19, 81–92 (2018).
[2] P. A. Boriack-Sjodin & K. K. Swinger, Biochemistry 55, 1557–1569
(2016).
[3] D. K. Liscombe, G. V. Louie & J. P. Noel, Nat. Prod. Rep. 29, 1238
(2012).
[4] M. Fontecave, M. Atta & E. Mulliez, Trends Biochem. Sci. 29, 243–
249 (2004).
[5] A.-W. Struck, M. L. Thompson, L. S. Wong & J. Micklefield,
ChemBioChem 13, 2642–2655 (2012).
̆
[6] C. Dalhoff, G. Lukinavičius, S. Klimasauskas & E. Weinhold, Nat.
Chem. Biol. 2, 31–32 (2006).
[7] R. Wang, W. Zheng, H. Yu, H. Deng & M. Luo, J. Am. Chem. Soc.
133, 7648–7651 (2011).
[8] K. Islam, et al., Proc. Natl. Acad. Sci. U. S. A. 110, 16778–16783
(2013).
[9] R. Wang, et al., J. Am. Chem. Soc. 135, 1048–1056 (2013).
[10] K. Islam, et al., J. Am. Chem. Soc. 134, 5909–5915 (2012).
[11] B. J. C. Law, A.-W. Struck, M. R. Bennett, B. Wilkinson & J.
Micklefield, Chem. Sci. 6, 2885–2892 (2015).
[12] C. Zhang, R. L. Weller, J. S. Thorson & S. R. Rajski, J. Am. Chem.
Soc. 128, 2760–2761 (2006).
[13] S. Singh, et al., Angew. Chem. Int. Ed. 53, 3965–3969 (2014).
[14] F. Muttach, N. Muthmann & A. Rentmeister, Org. Biomol. Chem.
15, 278–284 (2017).
[15] K. Hartstock, et al., Angew. Chem. Int. Ed. 57, 6342–6346 (2018).
[16] C. Sommer-Kamann, A. Fries, S. Mordhorst, J. N. Andexer & M.
Müller, Angew. Chem. Int. Ed. 56, 4033–4036 (2017).
[17] T. D. Huber, B. R. Johnson, J. Zhang & J. S. Thorson, Curr. Opin.
Biotechnol. 42, 189–197 (2016).
[18] J. Zhang & Y. G. Zheng, ACS Chem. Biol. 11, 583–597 (2016).
[19] C. Liao & F. P. Seebeck, Nat. Catal. 2, 696–701 (2019).
[20] S. Mordhorst, J. Siegrist, M. Müller, M. Richter & J. N. Andexer,
Angew. Chem. Int. Ed. 56, 4037–4041 (2017).
[21] J. Kim, et al., Nature 498, 123–126 (2013).
[22] R. T. Byrne, et al., Acta Crystallogr. D Biol. Crystallogr. 69, 1090–
1098 (2013).
[23] J. Kim, et al., Nucleic Acids Res. 43, 4602–4613 (2015).
[24] C. P. Ting, et al., Science 365, 280–284 (2019).
[25] B. J. C. Law, et al., Angew. Chem. Int. Ed. 55, 2683–2687 (2016).
[26] M. R. Bennett, et al., Angew. Chem. Int. Ed. 57, 10600–10604
(2018).
[27] P. Kast, M. Asif-Ullah, N. Jiang & D. Hilvert, Proc. Natl. Acad. Sci.
U. S. A. 93, 5043–5048 (1996).
[28] C. Grisostomi, P. Kast, R. Pulido, J. Huynh & D. Hilvert, Bioorganic
Chem. 25, 297–305 (1997).
[29] P. Ventosa-Andrés, L. Hradilová & V. Krchňák, ACS Comb. Sci. 16,
359–366 (2014).
[30] H. G. Floss & S. Lee, Acc. Chem. Res. 26, 116–122 (1993).
[31] J. Twilton, et al., Nat. Rev. Chem. 1, (2017).
[32] Z. Zuo, et al., Science 345, 437–440 (2014).
[33] C. P. Johnston, R. T. Smith, S. Allmendinger & D. W. C. MacMillan,
Nature 536, 322–325 (2016).
[34] E. R. Welin, C. Le, D. M. Arias-Rotondo, J. K. McCusker & D. W. C.
MacMillan, Science 355, 380–385 (2017).
[35] J. P. Phelan, et al., J. Am. Chem. Soc. 141, 3723–3732 (2019).
[36] C. Milne, et al., J. Am. Chem. Soc. 128, 11250–11259 (2006).
[37] C. Mahlert, F. Kopp, J. Thirlway, J. Micklefield & M. A. Marahiel, J.
Am. Chem. Soc. 129, 12011–12018 (2007).
[38] X.-W. Zou, et al., Acta Crystallogr. D Biol. Crystallogr. 70, 1549–
1560 (2014).
6
This article is protected by copyright. All rights reserved.

10.1002/anie.202004963
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
Methyltransferase enzymes (MT) deliver methyl substituents to an array of molecules with exquisite selectivity, controlling gene
expression or modulating the bioactivity of antibiotics and therapeutically important natural products. Here we show how MT can be
re-engineered for selective carboxy(m)ethylation and demonstrate how these orthogonal MTs can be combined with a SAM
derivatising enzyme, CmoA, to create new carboxymethylation pathways.
Institute and/or researcher Twitter usernames: @Micklefield_Lab & @UoMMIB
7
This article is protected by copyright. All rights reserved.