Expanding Radical SAM Chemistry by Using Radical Addition Reactions and SAM Analogues

Article abstract of DOI:10.1002/anie.201605917

Radical S-adenosyl-l-methionine (SAM) enzymes utilize a [4Fe-4S] cluster to bind SAM and reductively cleave its carbon–sulfur bond to produce a highly reactive 5′-deoxyadenosyl (dAdo) radical. In almost all cases, the dAdo radical abstracts a hydrogen atom from the substrates or from enzymes, thereby initiating a highly diverse array of reactions. Herein, we report a change of the dAdo radical-based chemistry from hydrogen abstraction to radical addition in the reaction of the radical SAM enzyme NosL. This change was achieved by using a substrate analogue containing an olefin moiety. We also showed that two SAM analogues containing different nucleoside functionalities initiate the radical-based reactions with high efficiencies. The radical adduct with the olefin produced in the reaction was found to undergo two divergent reactions, and the mechanistic insights into this process were investigated in detail. Our study demonstrates a promising strategy in expanding radical SAM chemistry, providing an effective way to access nucleoside-containing compounds by using radical SAM-dependent reactions.

Full text of DOI:10.1002/anie.201605917

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Communications
Chemie
International Edition: DOI: 10.1002/anie.201605917
German Edition: DOI: 10.1002/ange.201605917
Bioorganic Radicals
Expanding Radical SAM Chemistry by Using Radical Addition
Reactions and SAM Analogues
Xinjian Ji+, Yongzhen Li+, Liqi Xie, Haojie Lu, Wei Ding,* and Qi Zhang*
Abstract: Radical S-adenosyl-l-methionine (SAM) enzymes
utilize a [4Fe-4S] cluster to bind SAM and reductively cleave its
carbon–sulfur bond to produce a highly reactive 5’-deoxyade-
nosyl (dAdo) radical. In almost all cases, the dAdo radical
abstracts a hydrogen atom from the substrates or from
enzymes, thereby initiating a highly diverse array of reactions.
Herein, we report a change of the dAdo radical-based
chemistry from hydrogen abstraction to radical addition in
the reaction of the radical SAM enzyme NosL. This change
was achieved by using a substrate analogue containing an
olefin moiety. We also showed that two SAM analogues
containing different nucleoside functionalities initiate the
radical-based reactions with high efficiencies. The radical
adduct with the olefin produced in the reaction was found to
undergo two divergent reactions, and the mechanistic insights
into this process were investigated in detail. Our study
demonstrates a promising strategy in expanding radical SAM
chemistry, providing an effective way to access nucleoside-
containing compounds by using radical SAM-dependent
reactions.
has evolved to catalyze the addition of a dAdo radical to an
olefin (Figure 1A).[5] The rarity of the dAdo-radical-based
addition in radical SAM superfamily enzymes is in stark
contrast to the prevalence of radical addition reactions in
organic chemistry.[6] Herein, we report the change of the
T
he radical SAM superfamily represents the largest known
enzyme superfamily, consisting of more than 165000 mem-
bers found in all three domains of life.[1] These enzymes utilize
a [4Fe-4S] cluster to bind S-adenosyl-l-methionine (SAM) in
a bidentate fashion and reductively cleave its carbon-sulfur
bond to produce a highly reactive 5’-deoxyadenosyl (dAdo)
radical.[1b–e,2] Radical chemistry is then imposed on a variety
of organic substrates, leading to a highly diverse array of
biotransformations.[3] The dAdo radical can also be produced
Figure 1. Reactions catalyzed by the radical SAM enzymes MqnE and
NosL. A) The MqnE-catalyzed dAdo radical-based addition reaction
involved in menaquinone biosynthesis. B) The NosL-catalyzed promis-
cuous reactions with the authentic substrate l-Trp. C) Two routes of
hydrogen abstraction in NosL reaction with an unnatural substrate
MIPA. The two hydrogens abstracted by the dAdo radical are shown by
triangles and rectangles, respectively.
À
from adenosylcobalamin via C Co bond homolysis by the B12
radical superfamily enzymes.[4] For both radical SAM and B12
radical enzymes, the job of the dAdo radical is to abstract
a hydrogen atom from substrates or enzymes to initiate the
reaction. To date, the menaquinone biosynthesis enzyme
MqnE represents the only known radical SAM member that
dAdo-radical-based reaction of the enzyme NosL from
hydrogen abstraction to radical addition by using a double-
bond-containing substrate analogue. We show that NosL
accepts different SAM analogues with varied base moieties,
allowing effective production of different nucleoside adducts.
NosL is a radical SAM enzyme that catalyzes the carbon-
chain rearrangement of l-tryptophan (l-Trp) to produce 3-
methyl-2-indolic acid (MIA, 1; Figure 1B), a key intermedi-
ate in the biosynthesis of the clinically interesting thiopeptide
antibiotic nosiheptide.[7] This remarkable carbon-chain rear-
rangement reaction was recently suggested to proceed via
[*] X. Ji,[+] Prof. Dr. W. Ding
Ministry of Education Key Laboratory of Cell Activities and Stress
Adaptations
School of Life Sciences, Lanzhou University
Lanzhou, 730000 (China)
E-mail: dingw@lzu.edu.cn
X. Ji,[+] Dr. Y. Li,[+] Dr. L. Xie, Prof. Dr. H. Lu, Prof. Dr. Q. Zhang
Department of Chemistry, Fudan University
Shanghai, 200433 (China)
E-mail: qizhang@sioc.ac.cn
À
[+] These authors contributed equally to this work.
fragmentation of the l-Trp Ca C bond and subsequent
migration of the carboxyl-fragment radical to the indole C2
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under
(Figure 1B).[8] NosL also catalyzes the scission of the l-Trp
À
http://dx.doi.org/10.1002/anie.201605917.
Ca Cb bond to produce 3-methylindole (MI, 2) and glyox-
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ylate (Figure 1B).[7b] Both Ca C and Ca Cb fragmentations
of l-Trp result from the dAdo radical-mediated hydrogen
abstraction from the l-Trp amino group (Figure 1B).[9] We
recently showed that NosL is also able to produce MIA from
2-methyl-3-(indolyl)propanoic acid (MIPA), a Trp analogue
in which a methyl group replaces the amino group, and this
transformation is initiated by hydrogen abstraction from the
methyl group of MIPA (Figure 1C).[10] NosL also abstracts
a hydrogen from the MIPA Cb and produces a desaturated
product 2-methyl-3-(indolyl)acrylic acid (MIAA; Figure 1C),
demonstrating the remarkable catalytic promiscuity of this
enzyme.[10] Based on these observations, we reason that NosL
is likely able to recognize a MIPA structural analogue that
contains an olefin moiety, and in this case, the dAdo radical
would add to the olefin instead of initiation of the canonical
hydrogen abstraction process.
À
À
To test this hypothesis, we synthesized 2-(indolylmethyl)-
acrylic acid (IMAA, 3) and used it in a NosL assay (Fig-
ure 2A). Incubation of IMAA with SAM, sodium dithionite,
and the reconstituted NosL resulted in a product with
a protonated molecular ion at m/z = 453.1883 in liquid
Figure 3. NosL acts on two SAM analogues SGM and SCM. A) The
yields of different products in NosL reaction with SAM, SGM, or SCM.
Assays were carried out by incubating 500 mm substrates (l-Trp or
IMAA, 3) with 50 mm reconstituted protein, 500 mm SAM, and 2 mm of
sodium dithionite in 50 mm MOPS buffer (pH 8.0) for 1.5 h. Assays
were performed in duplicates, and the standard deviations (S.D.) are
shown by the error bars. B) Two types of reactions (hydrogen abstrac-
tion and radical addition) catalyzed by NosL with the SAM analogues.
characteristic product of the radical SAM superfamily
enzymes, was barely observable in the reaction (Figure S5),
suggesting that the dAdo radical was almost exclusively
trapped by IMAA rather than being reduced to dAdoH.
Despite the remarkable catalytic promiscuity of NosL-
catalyzed hydrogen abstraction reactions (Figure 1C),[10,11]
our analysis showed that NosL did not act on 2-(indolyl)-
acrylic acid (IAA), an IMAA analogue lacking a methylene
group of IMAA (Figure 2C).
Figure 2. Investigation of NosL reaction by using l-Trp analogues.
A) NosL-catalyzed reaction with IMAA 3, which produces an adenosine
adduct 4. B) LC-MS analysis of the IMAA reaction mixture, showing
the extracted ion chromatograms (EICs) of [M+H]+ =453.2 (corre-
sponding to 4) for i) NosL-catalyzed reaction with all the required
components, ii) control reaction with boiled NosL, and iii) control
reaction in which SAM was omitted. C) NosL does not act an IMAA
analogue 2-(indolyl)acrylic acid (IAA).
Unlike typical radical SAM chemistry, in which dAdo
radical only serves as a radical initiator, the dAdo-radical-
based addition reaction allows efficient adding of a nucleoside
moiety to the substrate. Production of the adenosine adduct 4
by NosL thus raises the tempting possibility of diversifying
radical SAM-dependent reactions by using SAM analogues
with different nucleotide functionalities. To test this hypoth-
esis, we synthesized S-guanosylmethionine (SGM), a SAM
structural analogue that has a guanine moiety (instead of an
adenine in SAM; Figure 3B). LC-HR-MS analysis of the
reaction mixture containing SGM, l-Trp, sodium dithionite,
and the reconstituted NosL showed that production of 5’-
deoxyguanosine (dGuoH), the cleavage product of SGM, was
indeed observed ([M+H]+ calc. 268.1046, obs. 268.1045,
0.4 ppm error). MIA (1) and MI (2) were also produced in
the reaction, and the yields of dGuoH, MIA, and MI were
comparable to those in the reaction with SAM (Figure 3A),
suggesting that SGM is a competent substrate for radical
initiation in NosL catalysis. Consistent with this observation,
incubation of SGM, IMAA, dithionite, and the reconstituted
NosL resulted in a guanosine adduct 5 (Figure 3B, [M+H]+
calc. 469.1836, obs. 469.1831, 1.1 ppm error), and the structure
is supported by comparative HR-MS/MS analysis (Figure S6).
These results demonstrate the possibility of expanding radical
chromatography (LC)/high-resolution mass spectrometry
(HR-MS) analysis (Figure 2B). The suggested molecule
formula C22H24N6O5 ([M+H]+ calc. 453.1886, 0.7 ppm error)
is consistent with an adenosine adduct of IMAA 4 (Fig-
ure 2A), and this is supported by detailed HR-MS/MS
analysis (Figure S1). In contrast to the low production yield
of MIA (1), production of 4 is efficient, with a yield more than
10-fold higher than that of MIA (Figure 3A). The relatively
high yield of 4 allowed recovering a sufficient amount of the
compound for NMR analysis (Figures S2–S3), which unequiv-
ocally showed that the dAdo radical-based addition proceeds
regiospecifically onto the C3, not C2, of IMAA (Figure 2A).
HPLC analysis using a prolonged elution program showed
that 4 likely consists of two diastereoisomers, and one
diastereoisomer (which likely has a 2R-configuration, see
below) accounts for approximately 75% of the total amount
(Figure S4). Production of 5’-deoxyadenosine (dAdoH), the
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SAM-dependent reactions by employing radical addition
that the majority of 4 likely has a (2R)-configuration (Fig-
reactions with a SAM analogue.
ure S9B). Notably, a small proportion ( ꢀ 12%) of di-deu-
terated 4 was observed in the assay mixture (Figure 4A). A
possible explanation is that the hydrogen abstraction in 4
production is a reversible event, which can also occur on other
sites of 4 (for example, the C5 of 4; Figure S8). Indeed,
detailed HR-MS/MS analysis of the di-deuterated 4 showed
that the two deuterium atoms are on the IMAA-derived
moiety, and part of the C5 of 4 was deuterated (Figure S10).
These results further highlighted the remarkable conforma-
tional diversity of NosL and the promiscuous substrate
binding in the enzyme active site.[10b]
Interestingly, we also observed a product that exhibits
a protonated molecular ion at m/z = 605.1848 in LC-HR-MS
analysis (Figure S11). Detailed HR-MS/MS analysis showed
an apparent neutral loss of m/z = 120. 02 (m/z = 470.13, and
350.11; Figures 4B and S11B) that is reminiscent of a dithio-
threitol (DTT) adduct observed in our previous study.[10a]
Indeed, the suggested molecule formula of C26H32N6O7S2
([M+H]+ calc. 605.1852, 0.7 ppm error) is consistent with 9,
a structural analogue of 4 containing an extra DTT moiety
(Figure 4B). Observation of 9 suggested that NosL produced
a desaturated product 8 in the assay (Figure 4B). Indeed,
examination of the LC-HR-MS data clearly showed that 8
was produced in the assay mixture, which exhibits a proton-
ated molecular ion consistent with the predicted molecular
formula C22H22N6O5 ([M+H]+ calc. 451.1730, obs. 451.1726,
0.9 ppm error; Figure S12). Production of the desaturated
product 8 suggested that besides being reduced by a solvent-
exchangeable hydrogen equivalent, the radical adduct 7 can
also undergo a one-electron-oxidation coupled with deproto-
nation of the C5 methylene group. The regio-specificity of this
desaturation process is likely a result of the relatively higher
activity of the C5 hydrogen compared to that of C3 (Fig-
ure 4B). The analogues of 8 and 9 that contain guanine or
cytosine were also observed in the reactions when SGM or
SCM were used (Figure S13).
We next performed the reaction with different concen-
tration of sodium dithionite. HPLC analysis of the resulting
assays showed that the yield of 4 increased with the increased
dithionite concentration (Figure S14), supporting the require-
ment of external electron donor to facilitate 4 production.
However, the production of 8 and 9 remains constantly low in
all of the dithionite concentrations, and was not observed
when the dithionite concentration was very high (> 5 mm). To
further interrogate the redox stoichiometry in 8 formation,
reconstituted NosL was incubated overnight with sodium
dithionite, allowing the [4Fe-4S] cluster to be fully reduced
and the excess dithionite to decompose via disproportiona-
tion, and the reduced enzyme was subsequently incubated
with SAM and IMAA in the absence of dithionite. This redox
stoichiometry analysis was developed by Liu and co-workers
in the study of DesII,[12] and was recently used in our lab in
studying the radical SAM-dependent amine dehydrogenation
reactions.[11] The UV/Vis spectrum of the reconstituted NosL
upon overnight reduction is similar to that of the freshly
reduced enzyme (Figure S15), supporting that the pre-
reduced enzyme was functional. LC-HR-MS analysis of the
reaction with the pre-reduced enzyme in the absence of
To further explore the substrate tolerance of NosL, we
synthesized another SAM analogue, S-cytidinylmethionine
(SCM), which contains a pyrimidine (instead of a purine in
SAM), therefore presenting a much bigger structural varia-
tion from SAM compared to SGM (Figure 3B). To test
whether SCM is able to initiate NosL reaction, SCM was
incubated in an assay mixture containing l-Trp, dithionite,
and the reconstituted NosL. HPLC and LC-HR-MS analysis
showed that MIA (1), MI (2), and 5’-deoxycytidine (dCydH)
were all produced in the assay, and the yields are slightly
decreased but comparable to those in the reaction with SAM
(Figure 3A). These results suggest NosL is able to effectively
cleave SCM and use a 5’-deoxycytidinyl radical to initiate the
reaction. Consistent with these results, a cytidine adduct 6 was
produced in the assay mixture containing SCM, IMAA, and
other required components (Figures 3A and S7). The com-
petence of SGM and SCM in the NosL reaction suggests that,
although conserved interactions are found in radical SAM
enzymes for binding of the adenine moiety of SAM,[2a] these
interactions are possibly not indispensable for SAM recog-
nition.
Unlike the reaction catalyzed by MqnE, in which the
dAdo radical adduct loses an electron and is decarboxylated
after the radical-mediated intermolecular rearrangement
(Figure 1A), the dAdo radical adduct with IMAA in NosL
reaction is reduced by a hydrogen equivalent. To reveal the
source of this hydrogen atom, we performed the assay with
IMAA in D2O buffer (pD 8.0, 90% D2O). LC-HR-MS
analysis of the resulting assay mixture showed that 4 is
predominately mono-deuterated (Figure 4A), suggesting that
the radical intermediate 7 is mainly quenched by a solvent-
exchangeable hydrogen equivalent, likely through hydrogen
abstraction from a solvent-exchangeable site (Figure S8).
Molecular docking analysis showed that the hydroxyl group
of NosL Tyr90 is close to the IMAA C2 (Figure S9A), which
could possibly provide a hydrogen source for 7 reduction. The
fact that Tyr90 resides at the Si face of IMAA also suggests
Figure 4. Mechanistic insights into the NosL-catalyzed dAdo radical-
based addition reaction. A) The mass spectrum of 4 produced in the
NosL reaction with IMAA in D2O buffer (pD 8.0, 90% D2O). The
spectrum clearly shows that 4 is mainly mono-deuterated and partially
di-deuterated. B) Divergence of the radical intermediate 7 into two
reaction pathways, producing the reduced product 4 and the desatu-
rated product 8, the latter is further converted to a DTT adduct 9 by
Michael addition of DTT to 8. Part of the collision-induced dissociation
(CID) ion fragments of 9 was shown, and the full HR-MS/MS
spectrum of 9 and 8 were shown in Figures S11B and S12B, respec-
tively.
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radical-based chemistry from hydrogen abstraction to radical
addition in NosL catalysis by using an olefin-containing
substrate analogue. Such a strategy could be well applied to
other radical SAM enzymes, as has also been shown in an
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Acknowledgements
This work supported in part by grants from the Fundamental
Research Funds for the Central Universities (lzujbky-2015-98
to W.D.), from National Natural Science Foundation of China
(1500028 to Q.Z.), and from the National Key Research and
Development Program (2016 Y F A0501302 to H.L. and
Q.Z.). Q.Z. would also like to thank the Thousand Talents
Program for support.
Keywords: S-adenosylmethionine · biosynthesis ·
radical addition · SAM analogues · thiopeptides
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Received: June 18, 2016
Revised: August 11, 2016
Published online: && &&, &&&&
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Communications
Bioorganic Radicals
X. Ji, Y. Li, L. Xie, H. Lu, W. Ding,*
Q. Zhang*
&&&& — &&&&
Expanding Radical SAM Chemistry by
Using Radical Addition Reactions and
SAM Analogues
SAM switch-up: The SAM-dependent
enzyme NosL was shown to switch from
hydrogen abstraction to radical addition
reaction when using an olefin-containing
substrate analogue. Two SAM analogues
with different nucleosides are able to
initiate the radical-based reactions com-
parable to SAM, offering a way to expand
SAM-dependent reactions and access
new nucleoside-containing compounds.
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