Substrate-Dependent Cleavage Site Selection by Unconventional Radical S-Adenosylmethionine Enzymes in Diphthamide Biosynthesis

Article abstract of DOI:10.1021/jacs.7b01712

S-Adenosylmethionine (SAM) has a sulfonium ion with three distinct C-S bonds. Conventional radical SAM enzymes use a [4Fe-4S] cluster to cleave homolytically the C_{5′,adenosine}-S bond of SAM to generate a 5′-deoxyadenosyl radical, which catalyzes various downstream chemical reactions. Radical SAM enzymes involved in diphthamide biosynthesis, such as Pyrococcus horikoshii Dph2 (PhDph2) and yeast Dph1-Dph2 instead cleave the C_γ,Met-S bond of methionine to generate a 3-amino-3-carboxylpropyl radical. We here show radical SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium sulfur by changing the structure of SAM. With a decarboxyl SAM analogue (dc-SAM), PhDph2 cleaves the C_methyl-S bond, forming 5′-deoxy-5′-(3-aminopropylthio) adenosine (dAPTA, 1). The methyl cleavage activity, like the cleavage of the other two C-S bonds, is dependent on the presence of a [4Fe-4S]⁺ cluster. Electron-nuclear double resonance and mass spectroscopy data suggests that mechanistically one of the S atoms in the [4Fe-4S] cluster captures the methyl group from dc-SAM, forming a distinct EPR-active intermediate, which can transfer the methyl group to nucleophiles such as dithiothreitol. This reveals the [4Fe-4S] cluster in a radical SAM enzyme can be tuned to cleave any one of the three bonds to the sulfonium sulfur of SAM or analogues, and is the first demonstration a radical SAM enzyme could switch from an Fe-based one electron transfer reaction to a S-based two electron transfer reaction in a substrate-dependent manner. This study provides an illustration of the versatile reactivity of Fe-S clusters.

Full text of DOI:10.1021/jacs.7b01712

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Communication
Substrate-Dependent Cleavage Site Selection by Unconventional
Radical SAM Enzymes in Diphthamide Biosynthesis
Min Dong, Masaki Horitani, Boris Dzikovski, Jack H. Freed, Steven Ealick, Brian M. Hoffman, and Hening Lin
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b01712 • Publication Date (Web): 06 Apr 2017
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Substrate-Dependent Cleavage Site Selection by Unconventional
Radical SAM Enzymes in Diphthamide Biosynthesis
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Min Dong,1 Masaki Horitani,2,3 Boris Dzikovski,1 Jack H. Freed,1 Steven E. Ealick,1 Brian M. Hoffman2
and Hening Lin4,*
1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, 14853. 2Department of Chemisꢀ
try, Northwestern University, Evanston, Illinois 60208. 3Department of Applied Biochemistry and Food Science, Saga Uniꢀ
versity, Saga 840ꢀ8502, Japan. 4 Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Cornell
University, Ithaca, New York, 14853
Supporting Information Placeholder
(ACP) radical, which is used in diphthamide biosynthesis in arꢀ
ABSTRACT: Sꢀadenosylmethionine (SAM) has a sulfonium ion
with three distinct CꢀS bonds. Conventional radical SAM enꢀ
chaeal and eukaryotic species.6 For example, the thermophilic
archaeon Pyrococcus horikoshii diphthamide biosynthesis protein,
PhDph2, transfers the ACP group from SAM to the substrate
protein, P. horikoshii translation elongation factor 2 (PhEF2).7,8
PhDph2 forms a homodimer and each monomer binds a [4Fe–4S]
cluster. The yeast Dph1ꢀDph2 heterodimer complex, an ortholog
of the PhDph2 homodimer, can also cleave SAM and transfer the
ACP group to yeast EF2 via a same radical mechanism.9
zymes use a [4Fe–4S] cluster to homolytically cleave the
C5′,adenosineꢀS bond of SAM to generate a 5′ꢀdeoxyadenosyl radiꢀ
cal, which catalyzes various downstream chemical reactions. Radꢀ
ical SAM enzymes involved in diphthamide biosynthesis, such as
Pyrococcus horikoshii Dph2 (PhDph2) and yeast Dph1ꢀDph2
instead cleave the Cγ,Met–S bond of methionine to generate the 3ꢀ
aminoꢀ3ꢀcarboxylpropyl radical. We here show that radical SAM
enzymes can be tuned to cleave the third CꢀS bond to the sulꢀ
fonium sulfur by changing the structure of SAM. With a decarꢀ
boxyl SAM analogue (dcꢀSAM), PhDph2 cleaves the Cmethyl–S
bond, forming 5′ꢀdeoxyꢀ5′ꢀ(3ꢀaminopropylthio) adenosine (dAPꢀ
TA, 1). The methyl cleavage activity, like the cleavage of the
other two CꢀS bonds, is dependent on the presence of a [4Feꢀ4S]+
cluster. Electronꢀnuclear double resonance (ENDOR) and mass
spectroscopy data suggests that mechanistically, one of the S atꢀ
oms in the [4Feꢀ4S] cluster captures the methyl group from dcꢀ
SAM, forming a distinct EPRꢀactive intermediate, which can
transfer the methyl group to nucleophiles such as dithiothreitol.
This reveals that the [4Feꢀ4S] cluster in a radical SAM enzyme
can be tuned to cleave any one of the three bonds to the sulfonium
sulfur of SAM or analogues, and is the first demonstration that a
radical SAM enzyme could switch from an Feꢀbased one electron
transfer reaction to a Sꢀbased two electron transfer reaction in a
substrateꢀdependent manner. This study thus provides a remarkaꢀ
ble illustration of the versatile reactivity of FeꢀS clusters.
Figure 1. Three categories of C–S bond cleavage reactions of
SAM.
SꢀAdenosylmethionine (SAM) is a versatile molecule in biology
(Figure 1).1,2 In addition to being a wellꢀknown methyl donor in
many biological reactions, SAM is cleaved homolytically to genꢀ
erate methionine and a 5′ꢀdeoxyadenosyl (5′ꢀdA) radical (Figure
1), which can initiate numerous reactions. Proteins that catalyze
these reactions are known as radical SAM enzymes3,4 and comꢀ
prise a superfamily of ~113,000 members5 that share a conserved
three cysteine motif (mostly CxxxCxxC) that binds a [4Fe–4S]
cluster. SAM can also be homolytically cleaved by unconventionꢀ
al radical SAM enzymes to generate a 3ꢀaminoꢀ3ꢀcarboxylpropyl
When SAM is used as a methyl donor, the C–S bond between
the methyl group and sulfonium (Cmethyl–S bond) normally is
cleaved heterolytically, through nucleophilic attack by the
acceptor atom, without involving a [4Fe–4S] cluster (Figure 1).
Although there are several methylases that transfer methyl group
from SAM to inert carbon atoms through generation of a 5′ꢀdA
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radical to initiate the reaction,10 no radical SAM enzyme has been
found to homolytically cleave the Cmethyl–S bond.
longstanding question in the radical SAM enzyme
standard MTA, was generated in the reaction. C) ESIꢀMS of the
isolated new peak in B. The [M+1]+ of the demethyl product 1
and the fragment peak can be detected.
A
community is how the radical SAM enzymes control which C–S
bond in SAM is cleaved. In this study we find that PhDph2
cleaves the CMethyl–S bond of a SAM analogue, decarboxyl SAM
(dcꢀSAM), forming 5′ꢀdeoxyꢀ5′ꢀ(3ꢀaminopropylthio) adenosine
(dAPTA, 1), thus demonstrating for the first time that radical
SAM enzymes can cleave all three sulfonium CꢀS bonds, and in
so doing providing important insight into how different enzymes
control the cleavage of SAM.
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The fact that without the carboxyl group of SAM, PhDph2 could
cleave a different CꢀS bond was very intriguing and surprising.
Mechanistically, this showed that the amino and carboxyl groups
in SAM are important for positioning SAM in the right orientation
in the active site of PhDph2 for the natural enzymatic reaction to
happen.
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Figure 2. Structure of dcꢀSAM and daꢀSAM
In conventional radical SAM enzymes, the amino and carboxyl
groups of SAM coordinate the unique iron in the cluster.11 The
interaction of SAM with the iron sulfur cluster is critical for
activity. We began this study by synthesizing two SAM analogues
to test whether the amino and carboxyl groups of SAM are also
important in PhDph2ꢀcatalyzed reaction: deamino SAM (daꢀ
SAM) and decarboxylꢀSAM (dcꢀSAM) (Figure 2). Although
SAM analogues with the adenine changed to other bases have
been used to study a radical SAM enzyme NosL,12 daꢀSAM and
dcꢀSAM had not been used. The daꢀSAM was not a substrate of
PhDph2; we could only detect nonꢀenzymatic decomposition of
daꢀSAM but not PhDph2ꢀcatalyzed cleavage (Figure S1). In
contrast, based on HPLC analysis, incubation of dcꢀSAM with
PhDph2 produced a new compound that was different from 5'ꢀ
deoxyꢀ5′ꢀmethylthioadenosine (MTA), the product generated in
the reaction with natural substrate SAM (Figure 3B). This
compound was only produced when PhDph2 and dithionite were
present, suggesting that it was an enzymatic reaction product and
required the reduced [4Feꢀ4S] cluster. The rate of this reaction
was comparable to that of the natural substrate SAM (Figure S2).
The new compound was isolated and characterized by
electrospray ionization mass spectrometry (ESIꢀMS) to be
dAPTA, 1, the demethyl product of dcꢀSAM (Figure 3C).
Figure 4. PhDph2 catalyzes demethylation reactions on SAM
analogues 2 and 3.
It is known that the amino and carboxyl groups of SAM are
crucial in conventional radical SAM enzymes.13 The bidentate
interaction of the amino and carboxyl group of SAM with the
unique iron in the [4Fe–4S] cluster facilitates the electron transfer
from the unique iron to the sulfonium of SAM resulting in the
reductive cleavage of SAM and the generation of the 5′ꢀdA
radical. We thus tested whether conventional radical SAM
enzymes would recognize dcꢀSAM or daꢀSAM. We incubated dcꢀ
SAM or daꢀSAM with BtrN,14,15 a radical SAM dehydrogenase
involved in the biosynthesis of antibiotic butirosin B. No dcꢀSAM
or daꢀSAM cleavage product was detected (Figure S3). The result
highlighted the different reactivities of PhDph2 and conventional
radical SAM enzymes, but also showed that the amino and
carboxyl groups of SAM are important for the reactivities of both
types of enzymes.
We further investigated whether the amino group in dcꢀSAM
was important for the methyl cleavage by PhDPh2. We
synthesized two other SAM analogues (2 and 3, Figure 4), with an
mꢀdimethylaminoꢀphenoxyl and phenoxyl groups substituting the
amino group of dcꢀSAM. When analogue 2 was incubated with
PhDph2, we detected the formation of the demethyl product
compound 4. Similarly, when analogue 3 was incubated with
PhDph2, the demethyl product 5 was also produced (Figure S4).
These results suggested that the amino group in dcꢀSAM is not
important for this novel cleavage activity.
To determine the fate of the cleaved methyl group from dcꢀ
13
SAM, we used
C
ꢀdcꢀSAM and 13C NMR to monitor the
methyl
reaction. A new peak around 15 ppm was detected in the reaction,
which was absent in the control reaction without dithionite
(Figure S5A). The chemical shift indicated a methylated thiol
group, likely resulting from the reaction with the DTT molecules
in the buffer. This was further confirmed with the detection of
Badan (6ꢀbromoacetylꢀ2ꢀdimethylaminonaphthalene)ꢀderivatized
methylated DTT by LCꢀMS (Figure S5B). When we carried out
the same reaction with PhDph2 and dcꢀSAM in the absence of
DTT, demethylation still occurred, but was much slower. We
Figure 3. The reaction of dcꢀSAM with PhDph2. A) PhDph2 can
convert dcꢀSAM to dAPTA, 1. B) Highꢀperformance liquid chroꢀ
matography analysis showing a new product, different with the
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hypothesized that proteins in the reaction could be the methyl
acceptor in the absence of DTT. To test this, we tried the reaction
with the natural substrate SAM, a SN2 displacement mechanism
occurred: a sulfur atom in the reduced [4Feꢀ4S] cluster attacked
the sulfonium center of dcꢀSAM, forming a Sꢀmethylꢀ[4Feꢀ4S]
intermediate. Other nucleophiles, such as the thiol groups on DTT
or nucleophilic residues on proteins, would subsequently attack
the Sꢀmethylꢀ[4Feꢀ4S] intermediate and complete the methyl
transfer reaction (Figure 7). This intermediate, could be compared
to the catalytic organometallic intermediate in pyruvate formate
lyaseꢀactivate enzyme (PFLꢀAE).17 Both intermediates are formed
by substrate (SAM or SAM analogue) cleavage with bonding of
the nascent radical/cleavage product to the [4Feꢀ4S] cluster, with
the difference being whether the Fe or the S atom in the cluster is
directly bonded.
14
of PhDph2 and
C
ꢀdcꢀSAM without DTT in the buffer.
methyl
14
C
ꢀdcꢀSAM was prepared in situ by using SAM
methyl
14
decarboxylase (SAMDC) and
C
ꢀSAM before adding
methyl
PhDph2. Interestingly, the SAM decarbxylase in the reaction was
strongly labeled (Figure 5). The labeling of SAM decarboxylase
was dependent on dithionite, suggesting that the methylation
event was dependent on an active PhDph2 with the reduced [4Feꢀ
4S]+ cluster. These results showed that both small molecules and
proteins could serve as the methyl acceptor in the reaction with
PhDph2 and dcꢀSAM.
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Figure 5. PhDph2 transfers 14Cꢀmethyl group to SAM decarboxꢀ
14
ylase (SAMDC) from
C
ꢀdcꢀSAM. The top panel shows the
methyl
Coomassie blueꢀstained gel; the bottom panel shows the autoradiꢀ
ography. All the reactions in lanes 1ꢀ3 contained PhDph2. The
presence of other reagents is indicated below each lane.
Figure 6. A) Xꢀband CW EPR spectra of Dph1ꢀDph2 in the abꢀ
sence and presence of dcꢀSAM at 12 K. B) 2D fieldꢀfrequency 35
GHz 13C Mims ENDOR of the 60 min sample of Dph1ꢀ2 with
To explore the mechanism and search for intermediates in the
methyltransfer reaction, we switched to the yeast ortholog Dph1ꢀ
Dph2 system because the enzyme was active at room temperature,
which facilitated experiments that were designed to capture
potential reaction intermediate. Similar to PhDph2, Dph1ꢀDph2
cleaved dcꢀSAM to generate dAPTA and methylated SAMDC
(Figure S6). We set up Dph1ꢀDph2 reaction with dcꢀSAM without
DTT, collected samples at different time points by manually
freezing the samples in liquid nitrogen, and examined them by
eletron paramagnetic resonance (EPR) spectroscopy. A new
species with g = [2.03, 1.98, 1.95] accumulated while the original
[4Feꢀ4S]+ signal diminished at longer time points (Figure 6A).
The signal broadened beyond detection at 35 K (Figure S7). The
gꢀvalues and temperature dependence of the new species indicated
that it involved the cluster, and was not an organic radical. This
new species dissappeared when we added DTT, which indicated it
was a reactive intermediate (Figure S8).
13
C
ꢀdc SAM. Experimental fields are as indicated. Condiꢀ
methyl
tions: MW frequency = 34.8 GHz, MW pulse length (π/2) = 50 ns,
τ = 500 ns and T = 2 K.
As a test of this mechanism, we reasoned that this intermediate
should produce methanethiol after denaturation of the protein and
FeꢀS cluster in acidic conditions. Indeed, we were able to detect
the formation of methanethiol in the reaction after denaturation of
the protein subjected to turnover, while no methanethiol was
detected in the control reaction without dithionite (Figure S9).
We further characterized this intermediate by preparing it with
13
C
ꢀdc SAM and using Mims pulsed electronꢀnuclear double
methyl
resonance (ENDOR) spectroscopy. A small, apparently nearꢀ
isotropic coupling, aiso ≈ 0.6 MHz, was detected (Figure 6B). Such
a small coupling indicated that the 13Cꢀmethyl group was in the
vicinity of the cluster but only weakly interacting with its spin. In
previous studies of PFLꢀAE, the nonlocal throughꢀspace coupling
constant is 0.33MHz for 13CꢀMe SAM,16 and the isotropic hyperꢀ
fine coupling constant of 13Cꢀcarboxyl SAM is 0.71 MHz.13 The
methyl group could have been transferred to a molecule adjacent,
but we propose that it bound to a sulfur atom of the [4Feꢀ4S]
cluster. Although without precedent, the spin density on such a
sulfur would be small, and would plausibly lead to the small
observed coupling for a cluster Sꢀmethyl. Such a species could
arise if, instead of the reduced cluster causing homolytic cleavage
Figure 7. Proposed mechanism of PhDph2 catalyzed methyl
transfer reaction with dcꢀSAM.
Computational studies suggest that the bidentate coordination
of the amino and carboxyl groups of SAM to the unique cluster
iron of conventional radical SAM enzymes is essential for the
generation of the 5′ꢀdA radical. Firstly, the sulfonium ion and
unique iron orbitals should have matching energies, which is
required for electron transfer between two atoms.18 Secondly, the
bidentate structure and the resulting conformation of SAM
explain the regioselectivity of C5′, Ade–S bond cleavage.19 PhDph2,
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representing a different type of radical SAM enzymes, cleaves the
Supporting Information
Experimental materials and methods, supporting figures. This
material is available free of charge on the ACS Publications webꢀ
site.
Cγ,Met–S bond in SAM and produce an ACP radical. Our results
here indicate that PhDph2ꢀcatalyzed reaction also requires both
the amino group and carboxylate group of SAM. However, unlike
classical radical SAM enzymes, PhDph2 can still accept the dcꢀ
SAM analogue as a substrate, but catalyzes the cleavage of the
CmethylꢀS bond, not the Cγ,MetꢀS bond cleaved in the natural
enzymatic reaction. Instead of a CxxxCxxC motif in traditional
radical SAM enzymes, the three conserved cysteine residues of
PhDph2 that bind the [4Fe–4S] cluster are located in separate
structural domains that separated by more than 100 residues in the
primary sequence.7 We believe this structure allows SAM to bind
the [4Feꢀ4S] in PhDph2 different from that in classical radical
SAM enzymes. The different binding mode of SAM in PhDph2
(and yeast Dph1ꢀDph2) likely contribute to the stereoelectronic
control of SAM cleavage and the unique reactivity with the SAM
analogues described here.
AUTHOR INFORMATION
Corresponding Author
Hening Lin: hl379@cornell.edu.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work is supported by NIH/NIGMS (GM 088276 and
P41GM103521 to HL and GM 111097 to BMH). We thank Dr.
Ivan Keresztes for assistance with NMR, Dr. Tyler L Grove and
Squire Booker at Penn State University for providing BtrN protein.
Although we cannot totally rule out the possibility of a radical
mechanism for formation of the Sꢀmethylꢀ[4Feꢀ4S] intermediate,
the SN2 nucleaphilic mechanism is most likely for the following
two reasons: (1) A homolytic cleavage of the CmethylꢀS bond in
SAM is difficult due to the high energy of methyl radical;4 (2) If a
methyl radical is generated, it should recombine with the closest
unique iron and form a CꢀFe bond, as the organometallic
intermediate in PFLꢀAE.17 Although recently the formation of low
level of 5'ꢀdeoxyꢀ5′ꢀthioadenosine, a reaction product of 5'ꢀ
deoxyadenosyl radical with one sulfur in the oxidized cluster, was
reported in NosL study. The iron sulfur cluster was inactivated
instead of playing a catalytic role.20 We therefore believe that this
is the first case in which a [4Feꢀ4S]+ cluster in a radical SAM
enzyme switches from catalyzing an Feꢀbased one electron transꢀ
fer reaction to catalyzing a Sꢀbased two electron transfer reaction
by simply changing the structure of SAM. This activity of
PhDph2 is dependent on the reduced cluster [4Feꢀ4S]+, which is
directly involved in the methyl transfer reaction.
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The methyl transfer studied here is different from that in the
traditional methyltransferase and other radical SAM enzymes with
methyl transfer activity. The wellꢀstudied methylsynthases RimN
and Cfr,21ꢀ23 use conserved cysteine residues to cleave the methyl
group from SAM via an SN2 mechanism. RimO and MiaB,24ꢀ26
representing a subgroup of methylthio transferase in radical SAM
enzyme family, consume two equivalent of SAM: one donates a
methyl group to the external sulfur attached to 4Feꢀ4S cluster,
another SAM is reductively cleaved to generate a 5'ꢀdA radical,
which abstracts a proton from the substrate to generate a substrate
radical. The substrate radical in turn attacks the methylthio group
on the 4Feꢀ4S cluster to complete the methylthio transfer. In our
case, PhDph2 or yeast Dph1ꢀDph2 only consumes one equivalent
of the SAM analogue in the catalytic cycle and the methyl accepꢀ
tor is a sulfur of the [4Feꢀ4S] cluster. A recent study reported that
NosN can use MTA, instead of SAM, as a direct methyl donor.27
The methyltransferase activity of PhDph2 on dcꢀSAM resembles
the cobalamin dependent SN2ꢀbased methyltransferase.28 Instead
of the reduced cobalt in methylcobalamin, the sulfur in [4Feꢀ4S]+
serves as an intermediate methyl acceptor to transfer methyl group
to other nucleophiles. Our studies here thus have revealed that the
[4Feꢀ4S] cluster in a radical SAM enzyme can be tuned to cleave
any one of the three bonds to the sulfonium sulfur of SAM or
analogues. Together with recent studies on PFLꢀAE and
PhDph2,17,29 our study provides compelling evidence for the verꢀ
satility of these [4Feꢀ4S] clusters.
(15) Grove, T. L.; Ahlum, J. H.; Sharma, P.; Krebs, C.; Booker, S. J.
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