Chemoenzymatic synthesis and in situ application of S-adenosyl-l-methionine analogs

Article abstract of DOI:10.1039/c3ob41702f

Analogs of S-adenosyl-l-methionine (SAM) are increasingly applied to the methyltransferase (MT) catalysed modification of biomolecules including proteins, nucleic acids, and small molecules. However, SAM and its analogs suffer from an inherent instability, and their chemical synthesis is challenged by low yields and difficulties in stereoisomer isolation and inhibition. Here we report the chemoenzymatic synthesis of a series of SAM analogs using wild-type (wt) and point mutants of two recently identified halogenases, SalL and FDAS. Molecular modelling studies are used to guide the rational design of mutants, and the enzymatic conversion of l-Met and other analogs into SAM analogs is demonstrated. We also apply this in situ enzymatic synthesis to the modification of a small peptide substrate by protein arginine methyltransferase 1 (PRMT1). This technique offers an attractive alternative to chemical synthesis and can be applied in situ to overcome stability and activity issues.

Full text of DOI:10.1039/c3ob41702f

Organic &
Biomolecular Chemistry
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Chemoenzymatic synthesis and in situ application of
S-adenosyl-L-methionine analogs†
Cite this: Org. Biomol. Chem., 2013, 11,
7606
Marie Thomsen,a,b Stine B. Vogensen,a Jens Buchardt,b Michael D. Burkart*c and
Rasmus P. Clausen*a
Analogs of S-adenosyl-L-methionine (SAM) are increasingly applied to the methyltransferase (MT) cata-
lysed modification of biomolecules including proteins, nucleic acids, and small molecules. However, SAM
and its analogs suffer from an inherent instability, and their chemical synthesis is challenged by low yields
and difficulties in stereoisomer isolation and inhibition. Here we report the chemoenzymatic synthesis of
a series of SAM analogs using wild-type (wt) and point mutants of two recently identified halogenases,
SalL and FDAS. Molecular modelling studies are used to guide the rational design of mutants, and the
enzymatic conversion of L-Met and other analogs into SAM analogs is demonstrated. We also apply this
in situ enzymatic synthesis to the modification of a small peptide substrate by protein arginine methyl-
transferase 1 (PRMT1). This technique offers an attractive alternative to chemical synthesis and can be
applied in situ to overcome stability and activity issues.
Received 20th August 2013,
Accepted 19th September 2013
DOI: 10.1039/c3ob41702f
www.rsc.org/obc
S-Adenosyl-L-methionine (SAM, 1a) is the most common respective MTases. This method offers a compelling strategy,
enzyme substrate in nature after ATP.1 It serves as a substrate as it allows for subsequent selective bioorthogonal conjugation
for methylation of proteins, nucleic acids, and small mole- of the MTase product with functional tags such as fluorescent
cules, but is also involved in polyamine biosynthesis and or affinity probes.3a,d–i
radical reactions.2 There has been considerable interest in
Currently, SAM analogs are synthesized chemically from
using analogs of SAM as selective inhibitors for exploring S-adenosyl-L-homocysteine (SAH, 2) with low yields and in an
methyltransferases (MTases) and for profiling MTase sub- approximately 1 : 1 ratio of the sulfonium ion diastereomers.
strates.3 The latter approach is highly interesting as it is specu- Usually only the (S,S) diastereomer is biologically active,
lated that many substrates are still to be identified.2b,4
whereas the (R,S) diastereomer has been demonstrated to
Two types of enzymatically active SAM analogs have been inhibit MTases.7 The impractical separation of these diastereo-
reported.5 Aziridinium-based SAM analogs have been mers typically leads to the application of diastereomeric mix-
employed with DNA and protein MTases resulting in bi-sub- tures for the MT transformations.3a, f–i Additionally, caution
strate products in which the SAM analog is covalently linked to must be applied when storing and handling SAM and SAM
the MTase substrate.3b,c,6 However, when using protein argi- analogs, as these molecules are inherently unstable, particu-
nine MTase 1 (PRMT1) the bi-substrate product has been larly at neutral and alkaline pH.8 An in situ enzymatic syn-
observed to cause product inhibition indicating that the scope thesis of SAM analogs would therefore be of high value if
of these analogs is limited.3c Recently, SAM analogs have been diastereoselective and able to overcome instability problems by
applied where the methyl group in L-Met of SAM is exchanged producing SAM analogs.
with a new reactive functional group (e.g. terminal alkynyl,
We recently demonstrated the enzymatic synthesis of SAM
keto, or amino group). Similar to the methyl group in SAM, the and its direct coupling to the enzymatic methylation of the
new chemical moiety is transferred to MTase substrates by the antibiotic teicoplanin by MtfA MTase and of DNA by HhaI
MTase.9 The applied SAM producing enzyme was a recently
discovered chlorinase SalL from Salinispora tropica10 and this
aDepartment of Drug Design and Pharmacology, Faculty of Health and Medical
Sciences, University of Copenhagen, Jagtvej 162, DK-2100, Copenhagen, Denmark.
E-mail: rac@sund.ku.dk
enzyme is homologous to a fluorinase FDAS from Streptomyces
cattleya.11 In vivo these halogenases catalyse the breakdown of
SAM by a nucleophilic attack of chloride or fluoride at C5′ of
the ribosyl moiety of SAM producing L-Met (3a) and 5′-chloro
or fluoro-5′-deoxyadenosine (ClDA or FDA, 4, X = Cl or F)
(Fig. 1). FDAS can also utilize chloride as a nucleophile, and
for both enzymes it has been shown that in vitro equilibrium
bDepartment of Biopharm Chemistry, Novo Nordisk A/S, 2760 Maaloev, Denmark
cDepartment of Chemistry and Biochemistry, University of California, San Diego,
CA 92093, USA
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ob41702f
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For FDAS, all mutants were expressed in sufficient yield
whereas for SalL, only 6 out of the 12 mutants were expressed
properly (T128A and Y239H-A) (ESI†). The activities of these
expressed mutants, as well as wt enzymes, were analyzed for
activity with L-Met (3a) and the series of synthetic L-Met
analogs (3b–j) of varying size and polarity, as illustrated in
Fig. 1 (see the tables with activity data in ESI†).
Activities were estimated by HPLC and reported in terms of
initial velocities from the ratio of the product area against the
total area of product and unreacted substrate (ClDA) relative to
the applied enzyme concentrations. Identical concentrations
(15 mM) of L-Met and its analogs were used except for benzyl
and phenethyl analogs (1.5 mM) due to low solubility. Negative
control reactions without enzymes were performed in each
series to verify enzyme activity. Consistent with earlier studies,
the activity of L-Met was approximately 500-fold higher for SalL
compared to FDAS.11,12d In general, activity with L-Met was
lower for the mutants compared to wt enzymes, except for
Y266FF and T155SF, which displayed a slightly increased
activity compared to wt FDAS.
Fig. 1 The enzymatic synthesis of SAM analogs and its coupling to the modifi-
cation of MTase substrates.
For wt SalL, activity was detected for all L-Met analogs
except propargyl (3f), phenethyl (3h) and polar analogs (3i,j)
(Fig. 3 and ESI†). The formation of ethyl-, propyl, butyl-, allyl-,
and benzyl-SAM was confirmed by LC MS (Fig. 3 and ESI†). In
general, activity decreased with increasing the size of the L-Met
favors the synthesis of SAM from ClDA and L-Met.10,12 Herein,
we report the successful use of wt SalL, designed SalL point
mutants, and FDAS for the enzymatic diastereoselective syn-
thesis of SAM analogs from ClDA and readily prepared L-Met
analogs (3b–j) (Fig. 1).
Inspecting molecular interaction field calculations of the
SalL (PDB: 2Q6I) and FDAS (PDB: 1RQR) crystal structures con-
taining L-Met,10,11 two water molecules were found tightly
bound in the active site of the enzymes where the L-Met
analogs are assumed to propagate (Fig. 2 and ESI†). This
suggests that destabilization of these water molecules might
facilitate the accommodation of larger substituents. When
analysing refined structures of SalL and FDAS, the following
amino acids were found to be involved in an extensive hydro-
gen bonding network with the two water molecules in SalL (S)
and FDAS (F): T128S (T155F), W190S (W217F), Y239S (Y266F),
backbone amide of S242S (S269F), and the carboxylate group of
L-Met (Fig. 2). A series of point mutants were generated for
this T, W and Y in both SalL and FDAS with the aim of accom-
modating larger substituents through the introduction of
smaller amino acids and destabilizing the hydrogen bonding
network (Fig. 2).
Fig. 3 (A) Enzyme activity of wt SalL and the W190 mutant series of SalL using
L-Met analogs. (B) HPLC chromatograms illustrating the formation of butyl-SAM
using wt and W190A SalL. The synthesis of butyl-SAM was verified by LC MS.
Mmono = monoisotopic mass. *1.5 mM S-benzyl-L-Hcy. ∇These peaks are impuri-
ties from the enzyme solutions.
Fig. 2 The active site of SalL overlaid with the molecular interaction field of
water at −10.0 kcal mol−1 (black surfaces) and listing of the series of mutations
for T128S (T155F), W190S (W217F) and Y239S (Y266F).
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analog. The rate of formation of ethyl-, propyl, butyl-, and allyl-
SAM compared to SAM was reduced 10-, 5000-, 15 000- and
5000-fold, respectively.
For some mutants the decrease in activity relative to L-Met
was much less pronounced (Fig. 3 and ESI†). In particular, the
Trp mutant series showed good activity. For W190FS, activity to
L-Met analogs decreased in a similar manner to that observed
for wt SalL. For W190HS, activity was observed using the
propyl and butyl analogs of L-Met, but no activity was detected
using either L-Met or the ethyl analog suggesting increased
activity towards the larger L-Met analogs. The W190AS mutant
accepted the same set of L-Met analogs as wt SalL, but the
activities were not reduced as drastically as seen for SalL and
the other mutants. Here, the activity of ethyl-, propyl, butyl-,
and allyl-SAM formation compared to SAM formation was
reduced 20-, 30-, 30-, and 60-fold, respectively. These results
indicate that the activity of the ethyl analog of L-Met is reduced
with a similar magnitude compared to L-Met for both wt and
W190A SalL, whereas the reduction in activity when using the
larger substrates was 150–500-fold higher for wt SalL. W190AS
catalyzed the formation of butyl-SAM with a three-fold higher
Fig. 4 The enzyme activity of wt and mutants of FDAS. Note that one y-axis is
for L-Met and the other is for the ethyl analog.
mutants, respectively. Activities of L-Met and the ethyl analog
of L-Met were observed for all FDAS variants and the relative
reductions in activity when exchanging L-Met with the ethyl
analog were comparable with those found for wt SalL. Further-
more, HPLC analysis indicated the formation of trace amounts
of propyl-SAM for Y266F, but this could not be confirmed by
LC MS because the produced amount was below the detection
limit.
To probe the application of these enzymes in an in situ
enzymatic synthesis of SAM and its analogs coupled with the
modification of various substrates by MTs, we applied rat
PRMT1 to explore the enzymatic modification of a small
peptide sequence, the RGG peptide, that mimics the natural
substrate fibrillarin (Fig. 5). PRMT1 is the predominant PRMT
velocity than when using wt SalL (0.934
0.010 nM min−1
compared to 0.318 0.006 nM min−1). Propyl-SAM was pro-
duced with a similar velocity as with wt SalL, whereas the for-
mation of SAM was reduced 160-fold compared to wt SalL.
Unlike wt SalL, W190AS showed no reduction in activity when
exchanging the propyl analog of L-Met with the butyl analog.
Only wt and W190A SalL were tested for activity towards the
benzyl and phenethyl analogs of L-Met, and similar rates for
the two enzymes were seen for the benzyl analog. This finding
is intriguing with respect to further development of enzyme
variants in order to discover even broader substrate specifici-
ties. From the Tyr mutant series, only Y239FS showed
sufficient expression yield to study the activity. This mutant
showed a similar reduction in activity to wt SalL when increas-
ing the size of the L-Met analogs. Finally, the Thr mutant
series showed activity only for L-Met and the ethyl analog of
L-Met. Unfortunately, the propargyl analog of L-Met was observed
to be unstable, but also propargyl-SAM has been reported to
be highly unstable (see ESI†).3e,f,h
These data partially confirm our in silico structural analysis
of SalL and FDAS, as we succeeded in modulating the active
site to accept some L-Met analogs. Mutants designed to reduce
the occupancy of water (W190FS and Y239FS) led to reduced
activity of all analogs, presumably due to impaired interaction
with the carboxylate group of L-Met and its analogs. But for
W190AS, this loss was compensated for by an increased size of
the pocket, leading to activities comparable to wt for propyl,
allyl, and benzyl L-Met analogs; and for the butyl analog a sig-
nificant increase in activity was observed. Wt and mutant
FDAS generally displayed much lower activities compared to
SalL (Fig. 4 and ESI†). However, two mutants (T155SF and
Y266FF) showed a small but significant increase in the activity
towards both L-Met and the ethyl analog of L-Met – ranging
from an L-Met activity of 10.6 1.5 nM min−1 for wt FDAS to
Fig. 5 (A) A schematic illustration of the coupling of the formation of enzy-
matic SAM and its analogs and the modification of the RGG peptide by rPRMT1.
(B) LC MS chromatogram of the coupled assay with the allyl analog of L-Met
and the spectra of the RGG peptide. L-MetX is an abbreviation for both L-Met
18.5 0.1 and 15.6 0.5 nM min−1 for the T155SF and Y266FF and its analogs.
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in mammalian cells, estimated to be responsible for 85% of
the arginine methylation.13 As a type 1 PRMT, the product of
Acknowledgements
PRMT1 is asymmetrically dimethylated arginine.14 First, we The authors thank Professor Bradley S. Moore and Professor
tested the use of the natural substrate SAM as well as the James H. Naismith for the kind donation of the plasmids
in situ SAM producing coupled assay using L-Met, ClDA, and pAEM7 and pFLA_HT for the expression of SalL and FDAS,
wt SalL. The peptide was successfully dimethylated at all three respectively. This work was financed by the Drug Research
arginines under both conditions (ESI†). Coupled assays Academy Program, the Novo Nordisk STAR program and NIH
were then performed using the ethyl, propyl, butyl, allyl, and R01GM095970.
benzyl analogs of L-Met, along with negative controls without
rPRMT1.
The allyl L-Met analog was transferred effectively, and both
mono- and di-modified peptides were identified by LC MS.
Notes and references
While allylation via synthetic analogs has been reported,14 this
is the first time that di-allylation has been seen using rPRMT1.
This finding is likely the result of our technique providing only
the biologically active (S,S) diastereomer and that no inhibitory
(R,S) diastereomer contaminates the allyl-SAM preparation.
Indeed, these findings strongly support the use of this chemo-
enzymatic method for a stereoselective analog preparation.
Transfer was also observed when using both the ethyl and
benzyl analogs of L-Met. It is notable that the bulky benzyl
group is transferred and this is probably due to the increased
reactivity of this group. For the benzyl analog, MS indicated
the formation of only a mono-modified peptide, whereas for
the ethyl analog mono-, di- and tri-modified RGG peptides
were identified.
However, when employing the larger alkyl-SAM analogs
(propyl and butyl), no activity was observed. Given that the
rate of propyl- and butyl-SAM formation is approximately 500-
fold lower when compared to ethyl-SAM, it remains unclear
whether this lack of activity is simply due to reduced specificity
or whether the activity is abolished.
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In conclusion, this study demonstrates the first published
method for chemoenzymatic synthesis of various SAM analogs
using both wt and engineered halogenases. This method uses
synthetically accessible L-Met analogs as precursors for the dia-
stereoselective formation of SAM analogs. The method is
useful for enzymatic modification by MTs using in situ gener-
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assays with other MTs becomes evident when considering the
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