Directed Evolution of a Halide Methyltransferase Enables Biocatalytic Synthesis of Diverse SAM Analogs

Article abstract of DOI:10.1002/anie.202013871

Biocatalytic alkylations are important reactions to obtain chemo-, regio- and stereoselectively alkylated compounds. This can be achieved using S-adenosyl-l-methionine (SAM)-dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide-based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl-, propyl-, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15 mg SAH). The V140T AtHMT was used in one-pot cascades with O-methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4-dihydroxybenzaldehyde. While a cascade for the propylation of 3,4-dihydroxybenzaldehyde gave low conversion, the propyl-SAH intermediate could be confirmed by NMR spectroscopy.

Full text of DOI:10.1002/anie.202013871

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Accepted Article
Title: Directed evolution of a halide methyltransferase enables
biocatalytic synthesis of diverse SAM analogues
Authors: Qingyun Tang, Christoph Grathwol, Aşkın S. Aslan-Üzel,
Shuke Wu, Andreas Link, Chris Badenhorst, Ioannis Pavlidis,
and Uwe Bornscheuer
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202013871
Link to VoR: https://doi.org/10.1002/anie.202013871

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Directed evolution of a halide methyltransferase enables
biocatalytic synthesis of diverse SAM analogues
Qingyun Tang,[a] Christoph W. Grathwol,[b] Aşkın S. Aslan-Üzel,[a] Shuke Wu,[a] Andreas Link,[b] Ioannis
V. Pavlidis*,[c] Christoffel P. S. Badenhorst*[a] and Uwe T. Bornscheuer*[a]
Abstract: Biocatalytic alkylations are important reactions to obtain
chemo-, regio- and stereoselectively alkylated compounds. This can
be achieved using S-adenosyl L-methionine (SAM)-dependent
methyltransferases and SAM analogues. It was recently shown that a
halide methyltransferase (HMT) from Chloracidobacterium
thermophilum can synthesize SAM from SAH and methyl iodide. We
developed an iodide-based assay for the directed evolution of an HMT
from Arabidopsis thaliana and used it to identify a V140T variant that
can also accept ethyl-, propyl-, and allyl iodide to produce the
corresponding SAM-analogues (90%, 50%, and 70% conversion of
15 mg SAH). The V140T-AtHMT was used in one-pot cascades with
O-methyltransferases (IeOMT or COMT) to achieve the regio-
selective ethylation of luteolin and allylation of 3,4-
dihydroxybenzaldehyde. While a cascade for the propylation of 3,4-
dihydroxybenzaldehyde gave low conversion, the propyl-SAH
intermediate could be confirmed by NMR spectroscopy.
MTs.[1c, 6] However, very few naturally occurring SAM analogues[5a,
6-7]
are known, making limited access to SAM analogues one of
the most serious impediments to progress in the field. With the
exception of S-adenosyl-L-ethionine (SAE), SAM analogues are
not readily available from commercial suppliers and have to be
prepared on demand. Alkylation of S-adenosyl L-homocysteine
(SAH) using alkyl halides results in low yields and contamination
with the biologically inactive (R,S) diastereomers.[5b] These side
products can be potent MT inhibitors and are not easily separated
from the desired SAM analogues.[8] Enzymatic synthesis of SAM
analogues is more specific and yields only the desired isomers.
Methionine adenosyltransferases (MATs) or halogenases
catalyze the production of SAM analogues by combining the
adenosyl moiety of ATP or 5′-chloro-5′-deoxyadenosine,
respectively, to the sulfur atom of methionine analogues.[9] The
major drawback of these approaches is that they require
methionine analogues, which are expensive if at all commercially
available (Figure S1).
The "magic methyl effect" refers to the ability of an appropriately
placed methyl group to dramatically alter the biological properties
of a compound.[1] This makes selective alkylation of molecules
highly desirable.[2] Bioalkylations are important because of the
exquisite chemo-, regio- and stereospecificity achievable using
enzymes.[2-3] In nature, selective methylation is catalyzed by
methyltransferases (MT, E.C. 2.1.1.X) that use S-adenosyl-
L-methionine (SAM, "nature's methyl iodide") as methyl donor.[4]
Interestingly, many methyltransferases are insensitive to the size
of the alkyl substituent of SAM and could catalyze other alkylation
reactions if the necessary SAM analogues were available.[5] This
promiscuity can be harnessed to expand the structural and
functional diversity of chemicals and enable various applications,
such as site-selective modification of molecules with fluorescent
or "clickable groups".[4a, 5a]
Liao and Seebeck recently reported the enzymatic synthesis
of SAM from SAH and methyl iodide, using
a halide
methyltransferase (HMT) from Chloracidobacterium
thermophilum (CtHMT).[10] We realized that, like many MTs, some
HMTs would be promiscuous and that this would allow a diverse
set of SAM analogues to be enzymatically synthesized using
cheap and readily available alkyl iodides, without having to
chemically synthesize methionine analogues.
We
expressed
and
purified
the
HMT
from
Chloracidobacterium thermophilum (CtHMT) originally described
by Liao and Seebeck.[10] We constructed an SAH nucleosidase-
deficient strain of E. coli BL21(DE3) to avoid contamination of
recombinant enzymes with this SAH-degrading enzyme
(Method S1.3). We also prepared HMTs from Arabidopsis
thaliana (AtHMT) and Raphanus sativus (RsHMT) as these have
been well characterized and have high activities towards
iodide.[11] We assessed the abilities of the three recombinant
HMTs to alkylate SAH, using methyl iodide (MeI), ethyl iodide (EtI),
propyl iodide (PrI), and butyl iodide (BuI) as alkyl donors. All three
HMTs were active using methyl iodide, although CtHMT was
significantly less active than AtHMT and RsHMT (Table S1). As
expected, all three enzymes had promiscuous activity against
ethyl iodide, although this was three orders of magnitude lower
than methyltransferase activity for each of the enzymes (Table
S1). However, none of the enzymes had significant activity
towards propyl iodide or butyl iodide (less than 0.01 µmol/min/mg).
AtHMT had the highest ethyltransferase activity (2.69 ± 0.15
µmol/min/mg) and a solved crystal structure and was therefore
chosen as the starting point for semi-rational protein engineering.
SAM analogues enabling these diverse alkylation reactions
are crucial not only for expanding the industrial relevance of
biocatalytic alkylation but also for discovering promiscuous
[a]
M.Sc. Q. Tang, M.Sc. A. S. Aslan-Üzel, Dr. S. Wu, Dr. C. P. S.
Badenhorst, Prof. Dr. U. T. Bornscheuer
Institute of Biochemistry, University of Greifswald
Felix-Hausdorff-Str. 4, 17489 Greifswald, Germany
Email: cps.badenhorst@uni-greifswald.de; uwe.bornscheuer@uni-
greifswald.de
[b]
[c]
Dr. C. W. Grathwol, Prof. Dr. A. Link
Institute of Pharmacy, University of Greifswald
Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany
Assist. Prof. Dr. I. V. Pavlidis
Dept. of Chemistry, University of Crete
Voutes University Campus, 70013 Heraklion, Greece
Email: ipavlidis@uoc.gr
We docked EtI, PrI, and BuI into the active site of the AtHMT
crystal structure (PDB ID: 3LCC) with bound SAH (Figure 1a).[12]
The alkyl-binding pocket is quite spacious and could
Supporting Information for this article is given via a link at the end of
the document.
1
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10.1002/anie.202013871
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accommodate even butyl iodide (in agreement with alkyl
promiscuity of many methyltransferases). The iodine atoms of all
docked substrates were superposed. We selected the residues
forming the alkyl-binding site (P20, V23, L27, W36, W47, Y139,
V140, C143, Y172 and R214) for further investigation. For each
of these positions, an NNK site-saturation library was created
using mutagenesis PCR. For each NNK library, 96 clones were
screened by incubating crude lysate with 1 mM SAH and 5 mM
ethyl iodide (Supporting Method S2). While the formation of SAE
could be monitored by HPLC, this would require 20 hours per
library, making it very tedious to investigate all randomized
residues. Therefore, we needed a high-throughput method for
determining the amount of product formed in each assay.
Because an iodide ion is released for each molecule of SAE
formed (Figure 1b), we initially considered using our recently
published ultrasensitive halide assay.[13] However, we were
concerned that background signals originating from chloride in
crude cell lysates would complicate the interpretation of results.
Therefore, we developed a modified assay that is insensitive to
chloride but highly sensitive to iodide.
We used
a
recombinant Curvularia inaequalis vanadium-
dependent chloroperoxidase (CiVCPO) to oxidize iodide to
hypoiodous acid (HOI). The HOI formed oxidizes the chromogen
3,3′,5,5′-tetramethylbenzidine (TMB), resulting in the formation of
blue color (Figure S2a).[14] In the process, iodide is released again,
so that one iodide anion can catalyze the oxidation of multiple
TMB molecules (Figure 1b). This results in very high sensitivity,
and only 1 µl of sample is required for the assay. The change in
absorbance at 570 nm is directly proportional to iodide
concentration in the range from 5 µM to 400 µM (Figure S3a).
Chloride concentrations up to 1 M result in only slight color
changes (Figure S2b). Most importantly, our assay is general and
suitable for monitoring the release of iodide from a range of alkyl
iodides. This enables both high-throughput screening of mutant
libraries and screening of HMT variants against various alkyl
iodides. Activities of HMTs determined using the iodide assay
were comparable to those determined using HPLC (Figure S3c),
demonstrating the validity and reliability of the assay.
The 10 NNK libraries at the selected positions were screened
and four hits (V23T, W36F, V140C, and V140T) with improved
activities relative to the wild-type AtHMT were identified
(Figure S4). Based on these hits, we constructed NNK libraries at
each other hit position, for a total of six additional libraries
(Table S2). Hits from screening these libraries were double-
mutant combinations of V23K/L/T, W36F, and V140C/T. Finally,
we constructed triple mutants based on the single and double
mutants. We expressed and purified the wild-type AtHMT and all
these variants and determined their specific activities towards
ethyl iodide (Figure 2). The specific activity of the most active
V140T variant (14.02 µmol/min/mg) towards ethyl iodide was
fivefold higher than that of the wild-type AtHMT
(2.69 µmol/min/mg).
a)
Y139
Y172
R214
V140
SAH
C143
W36
Alkyl iodides
P20
W47
V23
L27
b)
Ethyl transfer to SAH
NH2
NH2
N
N
N
N
N
N
-OOC
S
-OOC
S
N
N
O
O
HMT
+
+
NH3
NH3
1
2
OHOH
OHOH
I-
I
Figure 2. Specific activities of wild-type AtHMT and several single, double, and
triple mutants, determined using ethyl iodide as substrate. Data plotted are the
means, with standard deviation, of three independent measurements. The data
used for plotting this graph are shown in Table S3.
Iodide assay
I-
CiVCPO
I-
HOI
H2O2
TMB
TMBox
Kinetic analysis (Figures S5 and S6) showed that the activity
improvement relied on an increased kcat and not decreased Km for
ethyl iodide (Table 1). The kcat value for the V140T was 6-fold
higher than that for the wild type. The relative preference for ethyl
transfer was 36-fold higher for the V140T variant than for the wild-
type AtHMT.
H2O
Figure 1. a) The crystal structure of AtHMT and the modelled alkyl iodides in
the active site. The selected mutated sites, SAH and the alkyl iodides which
included MeI, EtI, PrI, and BuI are shown as sticks with elemental coloring. The
carbons of SAH, MeI, EtI, PrI and BuI are shown in white, grey, purple, yellow
and pink, respectively. b) Transfer of the ethyl group from ethyl iodide to SAH
(1) and the production of S-adenosyl ethionine (SAE, 2) and iodide. The iodide
produced can be detected using hydrogen peroxide, a haloperoxidase, and
TMB.
2
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Table 1. Kinetic parameters of wild-type AtHMT and the V140T variant.
Methyltransferase activity
Ethyltransferase activity
Substrate preference
[EtI]/[MeI][a]
Variant
kcat/Km, MeI
(min-1/mM)
kcat/Km, EtI
(min-1/mM)
kcat (min-1)
Km, MeI (mM)
kcat (min-1)
Km, EtI (mM)
WT
364.4 ± 12.7
182.4 ± 11.7
1.43 ± 0.19
4.70 ± 0.76
254.5
0.36 ± 0.03
2.38 ± 0.16
9.36 ± 1.66
0.038
0.214
1.5×10-4
5.5×10-3
V140T
38.7
11.14 ± 1.52
[a] Substrate preference for EtI relative to MeI is the ratio of kcat/Km values for the two substrates. This preference is ~36-fold higher for the V140T variant.
In addition to increased ethyltransferase activity, the V140T
variant was also more active towards propyl iodide and allyl iodide, maximize chances of finding improved variants, but the libraries
what you screen for′.[18] We screened using ethyl iodide to
compared to the wild type (Figure S7). The V140T variant was
used to convert SAH (15 mg) to SAE (2), S-propyl L-homocysteine
and screening methodology reported here could be used to
identify variants with improved activity towards other alkyl iodides.
(SAP (3)), and S-allyl L-homocysteine (SAA (4)) using ethyl iodide, Exploring the intrinsic alkyl-substituent promiscuity of naturally
propyl iodide, and allyl iodide. Conversions were 90%, 50%, and
70% after 14, 24, and 14 h, respectively (Figure 3a and Table S4).
The V140T variant is therefore valuable for the synthesis of
various SAM analogues. However, using stoichiometric amounts
of these alkylating agents would be expensive due to poor atom
economy and the high cost of SAH. Therefore, we used the
V140T AtHMT for in vitro regeneration of SAE, SAP, and SAA,
from catalytic amounts of SAH, in one-pot biocatalytic alkylation
cascades (Figure 3b). The T133M-Y326L variant of isoeugenol
O-MT (IeOMT) and the V140T-AtHMT catalyzed regioselective
mono-ethylation of luteolin to 3′-O-ethylluteolin (6) with 41%
conversion.[15] Similarly, human catechol O-MT (COMT) in
cascade with V140T-AtHMT catalyzed regioselective mono-
allylation of 3,4-dihydroxybenzaldehyde to produce 4-allyloxy-3-
hydroxybenz-aldehyde (8) with 48 % conversion (Table S5).[16]
The identities of the products were confirmed by 1H and 13C NMR
spectroscopy (Figures S12-S17). The conversions achieved
correspond to 41 and 48 SAH regeneration cycles. While a
cascade of human COMT and V140T-AtHMT catalyzed the
propylation of 3,4-dihydroxybenzaldehyde, the conversion was
low (~5%) and the product could not be purified for NMR
spectroscopy. Therefore, we isolated SAP synthesized using the
V140T-AtHMT (Figure S9b) and confirmed its structure by 1H and
13C NMR (Figures S18-S19).
occurring HMTs presents a very promising avenue for future
research. Not only might a highly promiscuous variant already
exist in nature, but also chances of finding an optimal starting
point for directed evolution would be improved by characterizing
a larger number of extant proteins.
a)
R
-OOC
S
HMT
-OOC
S
A
A
+
NH3
+
NH3
I-
R
I
R =
2, 90%
3, 50%
4, 70%
b)
MT
Compound
R
Alkylated Compound
-OOC
S
-OOC
S
A
A
+
NH3
+
NH3
We have demonstrated that the scope of biocatalytic
alkylations can be rapidly expanded by harnessing the
promiscuity of an engineered halide methyltransferase. The HMT
from Arabidopsis thaliana enabled us to produce three SAM
analogues from cheap and readily available alkyl iodides.
Importantly, we also demonstrated application of the engineered
HMT in bioalkylation cascades. It could catalyze over 40 cycles of
alkylation and SAH regeneration, allowing SAH to be used in
catalytic rather than stoichiometric amounts (Figure 3b). As noted
by Liao and Seebeck, the use of toxic alkyl iodides as reagents
might be considered a drawback of this approach. However, the
same applies to chemical alkylation and the biocatalytic
alternative functions under milder conditions and offers chemo-,
regio-, and stereoselectivity.[17]
I-
R
I
HMT
OH
O
O
OH
O
O
OH
OH
O
6, 41%, 41 cycles
8, 48%, 48 cycles
Figure 3. a) Preparative-scale (15 mg) synthesis of SAE (2), SAP (3), and
SAA (4) catalyzed by the V140T-AtHMT. The "A" represents the adenosyl
moiety. Conversions shown are from Table S4. b) Production (10 mg scale) of
alkylated products using cyclic MT-HMT cascades, employing 100 µM SAH and
80 mM alkyl iodide. The IeOMT variant T133M-Y326L and the V140T-AtHMT
catalyzed the ethylation of luteolin and produced 4′-O-ethylluteolin (6) with 41%
conversion. Human COMT and V140T-AtHMT catalyzed the allylation of
3,4-dihydroxybenzaldehyde and produced 4-allyloxy-3-hydroxybenzaldehyde
(8) with 48% conversion. Insignificant conversion took place if the HMT and MT
were not added (Figures S9 and S11). Conversions are from Table S5 and
Our best variant (V140T) had the highest activity against all
alkyl iodides tested (Figure S7), demonstrating that screening
using ethyl iodide can increase activity against other alkyl iodides
of interest. However, the ethyltransferase activity of V140T is
significantly higher than its propyltransferase activity, as ′you get
numbers of SAH regeneration cycles were calculated as [product]/[SAH]t = 0
.
3
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Z. Prokop, C. P. S. Badenhorst, U. T. Bornscheuer,
ChemCatChem 2020, 12, 2032-2039.
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Keywords: bioalkylation • protein engineering • halide
methyltransferase • SAM analogue • methylation
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4
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Entry for the Table of Contents
Biocatalytic alkylations are valuable for late-stage functionalization but are limited by availability of S-adenosyl methionine analogues.
We used directed evolution to create an engineered halide methyltransferase capable of converting cheap and readily available alkyl
iodides into a number of SAM analogues. Used in cascades with methyltransferases, this enables chemo-, regio- and stereoselective
alkylations hard to achieve by chemical means.
5
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