Asymmetric C-Alkylation by the S-Adenosylmethionine-Dependent Methyltransferase SgvM

Article abstract of DOI:10.1002/anie.201609375

S-Adenosylmethionine-dependent methyltransferases (MTs) play a decisive role in the biosynthesis of natural products and in epigenetic processes. MTs catalyze the methylation of heteroatoms and even of carbon atoms, which, in many cases, is a challenging reaction in conventional synthesis. However, C-MTs are often highly substrate-specific. Herein, we show that SgvM from Streptomyces griseoviridis features an extended substrate scope with respect to the nucleophile as well as the electrophile. Aside from its physiological substrate 4-methyl-2-oxovalerate, SgvM catalyzes the (di)methylation of pyruvate, 2-oxobutyrate, 2-oxovalerate, and phenylpyruvate at the β-carbon atom. Chiral-phase HPLC analysis revealed that the methylation of 2-oxovalerate occurs with R selectivity while the ethylation of 2-oxobutyrate with S-adenosylethionine results in the S enantiomer of 3-methyl-2-oxovalerate. Thus SgvM could be a valuable tool for asymmetric biocatalytic C-alkylation reactions.

Full text of DOI:10.1002/anie.201609375

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201609375
German Edition: DOI: 10.1002/ange.201609375
Biocatalysis
Asymmetric C-Alkylation by the S-Adenosylmethionine-Dependent
Methyltransferase SgvM
Christina Sommer-Kamann, Alexander Fries, Silja Mordhorst, Jennifer N. Andexer, and
Michael Mꢀller*
Dedicated to Professor Gerhard Bringmann on the occasion of his 65th birthday
Abstract: S-Adenosylmethionine-dependent methyltransfer-
ases (MTs) play a decisive role in the biosynthesis of natural
products and in epigenetic processes. MTs catalyze the
methylation of heteroatoms and even of carbon atoms,
which, in many cases, is a challenging reaction in conventional
synthesis. However, C-MTs are often highly substrate-specific.
Herein, we show that SgvM from Streptomyces griseoviridis
features an extended substrate scope with respect to the
nucleophile as well as the electrophile. Aside from its
physiological substrate 4-methyl-2-oxovalerate, SgvM cata-
lyzes the (di)methylation of pyruvate, 2-oxobutyrate, 2-oxo-
valerate, and phenylpyruvate at the b-carbon atom. Chiral-
phase HPLC analysis revealed that the methylation of
2-oxovalerate occurs with R selectivity while the ethylation of
2-oxobutyrate with S-adenosylethionine results in the S enan-
tiomer of 3-methyl-2-oxovalerate. Thus SgvM could be a val-
uable tool for asymmetric biocatalytic C-alkylation reactions.
been characterized in vitro.[8,9] This is due to the complexity of
radical SAM enzymes, which mainly stems from their oxygen
sensitivity and the need for an external electron source, and is
also due to the lack of knowledge regarding the physiological
methyl donor.
A few examples of asymmetric methylations catalyzed by
conventional SAM-dependent MTs, which follow an SN2-type
mechanism, have been described.[10] In the case of MT
domains of fungal polyketide synthases (PKSs), the MT
constitutes a distinct domain in the PKS that introduces
methyl groups stereoselectively at the growing carbon
chain.[11] However, this has been shown on the biosynthetic
level only.
The biosynthesis of some b-branched 2-oxo acids has been
described or proposed to proceed by MT-catalyzed (asym-
metric) C-methylation.[12–15] Specifically accepted substrates
include 2-oxoarginine, 2-oxoglutarate, indolepyruvate, and
phenylpyruvate. The resulting b-methylated 2-oxo acids serve
as precursors of antibiotics, such as 3-methylarginine, dapto-
mycin, indolmycin, maremycin, and mannopeptimycins.[12–15]
These enzymes usually only accept their physiological sub-
strate or their substrate range has not yet been determined.
Moreover, the stereocenter formed can be prone to racemi-
zation, as in the case of 3-methylindolepyruvate or
3-methylphenylpyruvate. In biosynthesis, this characteristic
is exploited by subsequent enzymatic transamination and
ketoreduction, resulting in the formation of vicinal stereo-
centers.[14,16] In most cases, the absolute configuration of the
stereocenter formed in the methylation step is not known.
Furthermore, enzymes catalyzing subsequent reaction steps
could also influence the initially formed stereocenter.
The MT SgvM from the biosynthetic gene cluster of the
octadepsipeptide antibiotic viridogrisein (produced by Strep-
tomyces griseoviridis) is known to catalyze the transfer of the
electrophilic methyl group of SAM to the C3 position of
4-methyl-2-oxovalerate (a-ketoleucine, 1). Afterwards, prod-
uct 2 is transaminated by the putative aminotransferase SgvG,
and the branched amino acid is subsequently incorporated
into viridogrisein (Scheme 1).[17] The amino acid sequence of
SgvM contains a specific binding motif for SAM, but no Fe–S
cluster binding motif (CXXCXXC), and is therefore de-
scribed herein as a conventional SAM-dependent MT.
We hypothesized that the biocatalytic application of
SAM-dependent MTs in the asymmetric methylation and
alkylation of nonphysiological substrates would be possible
in vitro. For this, we chose SgvM as the catalyst as the
T
he introduction of a methyl group is known to modulate the
physicochemical properties and biological activities of com-
pounds and can thus lead to an increase in activity of up to
several orders of magnitude.[1,2] If the methyl group is
attached to a stereocenter, an even higher potency boost
can arise.[1,3] Whereas the nonenzymatic methylation of
+
heteroatoms by electrophilic “CH3 ” synthons is straightfor-
À
ward, the stereoselective formation of C C bonds by
methylation is challenging and usually requires multistep
syntheses. In contrast, the asymmetric introduction of
a methyl group in natural product biosynthesis is performed
in a single step and catalyzed by methyltransferases (MTs),
which usually require S-adenosylmethionine (SAM) as the
methyl donor.[1,4,5]
Asymmetric enzymatic methylations are typically cata-
lyzed by “radical SAM” dependent MTs that utilize an iron–
sulfur cluster to generate a 5’-deoxyadenosine radical.[6,7]
However, only very few of the respective enzymes have
[*] C. Sommer-Kamann, Dr. A. Fries, S. Mordhorst,
Prof. Dr. J. N. Andexer, Prof. Dr. M. Mꢀller
Institut fꢀr Pharmazeutische Wissenschaften
Albert-Ludwigs-Universitꢁt Freiburg
Albertstrasse 25, 79104 Freiburg (Germany)
E-mail: michael.mueller@pharmazie.uni-freiburg.de
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201609375.
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Scheme 1. SgvM-catalyzed SAM-dependent methylation of a-ketoleu-
cine (1) yielding (R)-3-methylketoleucine [(R)-2], and its subsequent
incorporation into the octadepsipeptide antibiotic viridogrisein.
b-branched aliphatic products should be less prone to
racemization than their aromatic counterparts. In particular,
we aimed at increasing the range of both nucleophiles (2-oxo
acids) and electrophiles (derived from SAM derivatives) that
are accepted by SgvM. We anticipated that alkyl group
transfer would occur stereoselectively as the MTs MppJ,
MarI, and GlmT are known to catalyze the methylation of
their physiological substrates in an asymmetric fashion.[12–14]
The codon-optimized N-terminally His-tagged sgvM gene
was cloned into pET-28a(+), overexpressed in E. coli BL21-
Gold (DE3), and the protein was purified by Ni-NTA affinity
chromatography; 120 mg of pure SgvM per liter of culture
medium were obtained. The activity of SgvM was tested with
the physiological substrate, a-ketoleucine (1).[17] The cosub-
strate SAM was produced in situ from l-methionine and ATP
by the methionine adenosyltransferase from Thermococcus
kodakarensis (TkMAT), and the SAM-derived byproduct
S-adenosylhomocysteine (SAH) was degraded using methyl-
thioadenosine/SAH nucleosidase from E. coli (EcMTAN).[18]
In time-dependent LC-MS analysis, an increasing product
peak with the mass of the expected 3,4-dimethyl-2-oxovaler-
ate (2; m/z 142.7) was observed while the substrate peak
decreased accordingly. After 18 hours, the total conversion of
1 was determined (Scheme 1).[19]
This protocol was also used to investigate other substrates.
Thus the 2-oxo acids 2-oxovalerate (3), pyruvate (5),
2-oxobutyrate (6), and phenylpyruvate (8) were tested as
nonphysiological substrates (Scheme 2A–C). Furthermore,
l-leucine and l-norvaline (the corresponding amino acids of
1 and 3) and 2-oxoglutarate were tested. LC-MS analysis
confirmed the conversion of the 2-oxo acids 3, 5, 6, and 8. In
the case of 3 and 6, the reaction was complete after 18 hours; 8
gave a conversion of 80% while the conversion of 5 amounted
to only 10%. The chromatographic and spectroscopic proper-
ties of the enzymatic products 3-methyl-2-oxovalerate (4) and
3-methyl-2-oxobutyrate (7) were identical to those of authen-
tic reference substances. 2-Oxoglutarate, l-leucine, and
l-norvaline were not accepted by SgvM. The negative result
with the tested amino acids indicates that the 2-oxo acid
moiety of the substrates is most likely essential for substrate
recognition by SgvM.
Scheme 2. SgvM-catalyzed mono- and dialkylations with nonphysiolog-
ical substrates. Assays were incubated at 258C for 18 h (exception:
ethylation assay of 6 for 27 h). Reaction conditions: SgvM
(0.8 mgmLÀ1), TkMAT (0.5 mgmLÀ1), EcMTAN (0.01 mgmLÀ1), MT
substrate (1 mm), l-methionine/[13C-methyl]-l-methionine/l-ethionine
(1.5 mm), ATP (1.5 mm), assay buffer (50 mm Tris-HCl, 50 mm MgCl2,
20 mm KCl, pH 8.0). Conversion of 5 into 3 (F) below the detection
limit.
with the same m/z value as 3-methylphenylpyruvate (9),
demonstrating that SgvM can also methylate 2-oxo acids with
an aromatic residue (Scheme 2C).
The position of the methylation was confirmed with
13CH3-labeled methionine as the methyl donor, using 3 and 5
as test substrates. The enzymatic reactions were performed as
described above and analyzed by 13C NMR spectroscopy.
Comparison of the spectrum of the product obtained from 3
with that of authentic, unlabeled 4 showed that the methyl
group of SAM was introduced at the C3 atom of 3 (> 95%
conv., Scheme 2D). The reaction of pyruvate (5) with 13CH3-
labeled methionine catalyzed by SgvM resulted in mono- and
dimethylation to labeled 6 and 7 (see below).
To elucidate whether SgvM acts stereoselectively, the
enzymatic product 4 was analyzed by HPLC analysis on
a chiral stationary phase after derivatization with ortho-
phenylenediamine (see the Supporting Information).[20,21]
Comparison with authentic (R)- and (S)-4 indicated that the
main product had the R configuration (Scheme 2A and
Figure 1). Samples were analyzed after an incubation time
of 4, 16, and 27 hours, and the enantiomeric excess (ee) of the
enzymatic product (R)-4 was determined to be 97.5, 96, and
91% ee, respectively. As a reference, authentic samples of
(R)-4 and (S)-4 were separately dissolved in assay buffer and
incubated as for the enzymatic assay for 18 hours. A decrease
in enantiopurity from 92 to 87% ee was observed for the
R enantiomer, and from 85 to 78% ee in the case of the
S enantiomer. This suggests that the decrease in enantiopurity
observed in the enzymatic methylation assays is due to
nonenzymatic racemization.
Incubation of SgvM with pyruvate (5) and SAM gave two
products with identical retention times and m/z values as
compounds 6 and 7. Hence, pyruvate is methylated twice by
SgvM: the first methylation gives 6, which serves as the
second substrate and is methylated to provide 7 (Scheme 2B).
Incubation of 8 with SgvM and SAM resulted in a new peak
2
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Figure 1. A) rac-4. SgvM-catalyzed methylation of 3 and ethylation of 6
resulting in the R and S enantiomer of 4, respectively. B) HPLC-UV
(230 nm) traces of enzymatic assays after derivatization with ortho-
phenylenediamine obtained with a Chiral-OM column (5 mm,
250ꢂ4.6 mm) and hexane/2-propanol (98:2). Top: rac-4. Middle:
Methylation assay of 3 after incubation for 16 h, yielding (R)-4 (96%
ee). Bottom: Ethylation assay of 6 after incubation for 27 h, yielding
(S)-4.
Scheme 3. A) SgvM-catalyzed 13C-monomethylation of 2-oxobutyrate
(6). SgvM-catalyzed 13C-dimethylation of B) pyruvate (5), C) [2-13C]-5,
and D) [2,3-13C2]-5. “C” denotes 13C. Assays were incubated at 258C for
18 h. See Scheme 2 for the reaction conditions.
We expected that SgvM might also catalyze the transfer of
alkyl moieties other than methyl groups, as has already been
shown for other MTs.[22–24] To test this hypothesis, 2-oxobu-
tyrate (6) and SgvM were incubated with S-adenosylethionine
(derived in situ from l-ethionine) according to the standard
procedure (ca. 10% conversion). Using the same chiral-phase
HPLC method as described above, an excess of (S)-3-methyl-
2-oxovalerate [(S)-4] was detected (Figure 1 and Sche-
of a methyl group from SAM to the b-carbon atom of the
substrates is stereoselective. (R)-3-Methyl-2-oxovalerate
[(R)-4] was synthesized from 2-oxovalerate (3) and SAM.
Notably, SgvM also catalyzes stereoselective ethylation
reactions. (S)-4 was enantioselectively obtained by the ethyl-
ation of 6 using S-adenosylethionine as the electrophile.
Furthermore, the enzyme can conduct multiple alkylations of
a substrate. With 13C-labeled substrates, selectively labeled
chiral compounds or products with isotopically engendered
chirality are readily available.
me 2E).[25] Hence, SgvM-catalyzed asymmetric C C bond
À
formation by methylation of 3 and ethylation of 6 provides an
enantiocomplementary access to both enantiomers of 4 with
the same enzyme. Accordingly, substrates 1, 3, 5, and 8 were
successfully (di)ethylated, with both 3 and 5 yielding 3-ethyl-
2-oxovalerate (10, Scheme 2F; 30% conversion of 3, detected
by LC-MS; see the Supporting Information).
Hence, asymmetric alkylations by conventional SAM-
dependent enzymes, as demonstrated for SgvM, provide
a multitude of possible applications in biocatalysis. This
approach has potential for the enzymatic transfer of other
groups, such as phenyl or allyl moieties,[22] or for the
a-alkylation of enolizable carbonyl or carboxyl groups in
general. Whilst the common application of SAM-dependent
alkylations is limited owing to the high cost of SAM, this
drawback could be overcome by the use of a SAM regener-
ation system, as has been developed recently.[32] Moreover,
follow-up transformations, such as reductions, transamina-
tions, or thiamin diphosphate dependent decarboxylations
Based on the SgvM-catalyzed (di)alkylation of 5 and 6, we
considered convergent syntheses of isotopically labeled
a-ketovaline (7). These transformations were analyzed by
13C NMR spectroscopy (see the Supporting Information).
Incubation of 13CH3-labeled methionine and the unlabeled
2-oxo acids 6 (> 95% conv.) or 5 (ca. 10% conv.; Scheme 3A
and B) or the differently 13C-labeled pyruvates 5 (ca. 10%
conv.; Scheme 3C and D) with SgvM resulted in four
isotopologues of 7. The products contained up to four
selectively labeled carbon atoms. This approach provides
a new strategy for the synthesis of isotopically labeled
precursors used in the NMR spectroscopy of high-molecu-
lar-weight proteins.[26–29] Product [5-13C]-7, obtained from the
conversion of 6 with 13CH3-labeled methionine, is a compound
with isotopically engendered chirality (Scheme 3A).[30,31]
In summary, we have shown that the SAM-dependent
methyltransferase SgvM from S. griseoviridis features a broad
substrate and reaction scope. Aside from the physiological
substrate 4-methyl-2-oxovalerate (1), SgvM accepts other
aliphatic and aromatic 2-oxo acids as substrates. The transfer
and C C bond formations,[33,34] expand the possible use of the
À
generated chiral b-branched a-keto acids.
Acknowledgements
We thank Sascha Ferlaino for measuring the NMR spectra.
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Keywords: asymmetric methylation · biocatalysis ·
enzyme catalysis · ethylation · isotopes
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Manuscript received: September 24, 2016
Revised: November 13, 2016
Final Article published: && &&, &&&&
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Biocatalysis
C. Sommer-Kamann, A. Fries,
S. Mordhorst, J. N. Andexer,
M. Mꢁller*
&&&& — &&&&
Asymmetric C-Alkylation by the S-
Adenosylmethionine-Dependent
Methyltransferase SgvM
Enzymatic alkylation: The methyltrans-
ferase SgvM from Streptomyces griseovir-
idis has a broad substrate scope with
respect to both the nucleophilic and
electrophilic reaction partner. It can thus
catalyze the asymmetric methylation and
ethylation of a variety of a-oxo acids.
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