Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase

Article abstract of DOI:10.1021/jacs.5b11035

RimO is a member of the growing radical S-adenosylmethionine (SAM) superfamily of enzymes, which use a reduced [4Fe-4S] cluster to effect reductive cleavage of the 5′ C-S bond of SAM to form a 5′-deoxyadenosyl 5′-radical (5′-dA? intermediate. RimO uses this potent oxidant to catalyze the attachment of a methylthio group (-SCH₃) to C3 of aspartate 89 of protein S12, one of 21 proteins that compose the 30S subunit of the bacterial ribosome. However, the exact mechanism by which this transformation takes place has remained elusive. Herein, we describe the stereochemical course of the RimO reaction. Using peptide mimics of the S12 protein bearing deuterium at the 3 pro-R or 3 pro-S positions of the target aspartyl residue, we show that RimO from Bacteroides thetaiotaomicron (Bt) catalyzes abstraction of the pro-S hydrogen atom, as evidenced by the transfer of deuterium into 5′-deoxyadenosine (5′-dAH). The observed kinetic isotope effect on H atom versus D atom abstraction is ～1.9, suggesting that this step is at least partially rate determining. We also demonstrate that Bt RimO can utilize the flavodoxin/flavodoxin oxidoreductase/NADPH reducing system from Escherichia coli as a source of requisite electrons. Use of this in vivo reducing system decreases, but does not eliminate, formation of 5′-dAH in excess of methylthiolated product.

Full text of DOI:10.1021/jacs.5b11035

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Communication
The Stereochemical Course of the Reaction Catalyzed
by RimO, a Radical SAM Methylthiotransferase
Bradley J. Landgraf, and Squire J. Booker
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b11035 • Publication Date (Web): 12 Feb 2016
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The Stereochemical Course of the Reaction Catalyzed by
RimO, a Radical SAM Methylthiotransferase
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Bradley J. Landgraf 1 & Squire J. Booker 1,2,3
1 Department of Chemistry, 2Department of Biochemistry and Molecular Biology, and 3The Howard Hughes Mediꢀ
cal Institute, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
Supporting Information Placeholder
ABSTRACT: RimO is a member of the growing radical Sꢀ
adenosylmethionine (SAM) superfamily of enzymes, which
use a reduced [4Feꢀ4S] cluster to effect reductive cleavage of
the 5' CꢀS bond of SAM to form a 5'ꢀdeoxyadenosyl 5’ꢀ
radical (5'ꢀdA•) intermediate. RimO uses this potent oxiꢀ
dant to catalyze the attachment of a methylthio group (–
SCH3) to C3 of aspartate 89 of protein S12, one of 21 proꢀ
teins that compose the 30S subunit of the bacterial riboꢀ
some. However, the exact mechanism by which this transꢀ
formation takes place has remained elusive. Herein, we
Scheme 1. The reaction catalyzed by RimO, with the absoꢀ
lute configuration of the carbon atom to which the methylꢀ
thio group is appended indicated in green as 3R.
describe the stereochemical course of the RimO reaction.
As a member of the radical SAM (RS) superfamily of
Using peptide mimics of the S12 protein bearing deuterium
enzymes, RimO uses a [4Feꢀ4S]+ cluster to promote the
at the 3 pro-R or 3 pro-S positions of the target aspartyl
residue, we show that RimO from Bacteroides thetaiota-
omicron (Bt) catalyzes abstraction of the pro-S hydrogen
reductive cleavage of the 5’ CꢀS bond of SAM to form a 5’ꢀ
deoxyadenosyl 5’ꢀradical (5’ꢀdA•).5ꢀ7 This potent oxidant has
been suggested to abstract a hydrogen atom (H•) from C3
atom, as evidenced by the transfer of deuterium into 5’ꢀ
of D89 to activate it for methylthiolation, although this
deoxyadenosine (5’ꢀdAH). The observed kinetic isotope
particular step of the reaction has never been
effect on Hꢀatom versus Dꢀatom abstraction is ~1.9, sugꢀ
demonstrated.1,6,7 In addition to the [4Feꢀ4S] cluster that
participates in the reductive cleavage of SAM (RS cluster),
RimO harbors an additional [4Feꢀ4S] cluster (auxiliary
cluster) in its Nꢀterminal region. The auxiliary cluster was
gesting that this step is at least partially rate determining.
We also demonstrate that Bt RimO can utilize the flavodoxꢀ
in/flavodoxin oxidoreductase/NADPH reducing system
from Escherichia coli as a source of requisite electrons. Use
previously believed to be sacrificed during the first phase of
of this in vivo reducing system decreases—but does not
the reaction to provide the inserted sulfur atom6 in a
eliminate—formation of 5’ꢀdAH in excess of methylthiolated
mechanism analogous to those proposed for the RS
product.
sulfurtransferases, lipoyl synthase (LipA)8ꢀ12 and biotin
synthase (BioB).13ꢀ17 In the second phase of catalysis, the
RimO (ribosomal modification O) catalyzes the postꢀ
inserted sulfur atom was proposed to undergo methylation
translational modification of aspartate 89 (D89) of protein
by a canonical SAMꢀdependent SN2 mechanism.6 Recent
S12 to give 3ꢀmethylthioaspartate (3ꢀMSꢀD89).1 Protein S12
studies suggest, however, that RimO, and the related
enzyme, MiaB, catalyze the initial synthesis of a methylthio
group that is most likely attached externally to the auxiliary
Fe/S cluster, and that the entire methylthio group is
is a component of the 30S subunit of the bacterial ribosome,
and the loop on which D89 resides projects into the
acceptor site where aminoacyl tRNAs bind.2 The
modification itself is not essential, but Escherichia coli (Ec)
subsequently transferred intact to C3 of the aspartyl residue
that are capable of catalyzing this methylthiolation reaction
via radical chemistry.18,19 These observations indicate that
have a slight growth advantage over those that are not. This
RimO and MiaB are members of an emerging subclass of RS
growth advantage is believed to be related to enhanced
enzymes that, within a single active site, activate SAM both
translational fidelity.1 Two recent Xꢀray structures of the
for methyltransfer and for generation of a 5’ꢀdA•.20,21
Thermotoga maritima (2.3ꢀ2.5 Å) and Ec (2.4 Å) ribosome
Although RimOs both from Escherichia coli (Ec)6 and
at high resolution showed electron density for 3ꢀMSꢀD89,
from Thermotoga maritima (Tm)7 have been characterized,
and in the Ec structure the absolute stereochemistry at C3
Tm RimO is better behaved and exhibits significantly greatꢀ
er turnover. However, the need to use the artificial reductꢀ
ant, dithionite, in activity determinations of Tm RimO, inꢀ
was observed to be R.3,4 The methylthio group points toꢀ
wards the 6ꢀoxo group of N7ꢀmethylguanosine 527, a modiꢀ
fied nucleobase in 16S rRNA; however, the purpose of the
duces production of SAH and 5’ꢀdA that was believed to be
interaction between the modified protein residue and the
uncoupled from product formation. In this study, we show
nucleobase is unknown.
that RimO from the gut bacterium, Bacteroides thetaiota-
omicron, can utilize the Ec flavodoxin/flavodoxin oxidoreꢀ
ductase/NADPH (Fld/FldR/NADPH) reducing system as a
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source of electrons for catalysis. We also determine the
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stereochemistry of H• abstraction from D89 of the S12 proꢀ
tein through the use of chemoenzymatically synthesized
deuterated isotopomers at C3 of an aspartate residue incorꢀ
porated into a 13ꢀamino acid peptide mimic (13ꢀmer) of the
S12 protein. We observe transfer of deuterium into 5’ꢀ
deoxyadenosine only from the 13ꢀmer containing [(2S,3S)ꢀ
2,3ꢀ2H2]ꢀaspartate and not [(3R)ꢀ3ꢀ2H1]ꢀaspartate, indicatꢀ
ing that H• abstraction is indeed stereoselective, and that
insertion of the ꢀSCH3 group occurs with inversion of conꢀ
figuration at C3. The apparent kinetic isotope effect associꢀ
ated with Hꢀ versus Dꢀatom abstraction by the 5'ꢀdA• is
~1.9, indicating that Hꢀatom abstraction is at least partially
rateꢀlimiting.
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In previous studies of Ec and Tm RimOs, the chemical
reductant, dithionite, was used as the source of requisite
electrons for catalysis. Tm RimO catalyzed methylthiolation
in the presence of dithionite, but not in the presence of the
Ec Fld/FldR/NADPH reducing system, while Ec RimO did
not exhibit appreciable activity with either reductant. The
use of the chemical reductant in the Tm RimO reaction,
however, was believed to induce abortive cleavage of SAM,
as evidenced by production of 5’ꢀdAH and SAH that were in
excess over the methylthiolated product.7,18,19 The Tm geꢀ
nome does not appear to encode flavodoxins, but does enꢀ
code five ferredoxins, one or more of which might function
to deliver electrons to RS enzymes. However, because of our
interest in RS enzymes from gut microbiota and the desire
to work with enzymes that function optimally closer to amꢀ
bient temperature, we chose to study RimO from the major
human gut bacterium, Bacteroides thetaiotaomicron (Bt),
which does contain an annotated flavodoxin gene. Bt RimO
is 35% identical to Ec RimO and 39% identical to Tm RimO,
while Bt flavodoxin is 37% identical to Ec flavodoxin. The
gene encoding Bt RimO was coꢀexpressed with plasmid
pDB1282, and its protein product was isolated as previously
described for Tm and Ec RimOs.6,19
When Bt RimO was incubated under turnover condiꢀ
tions in the presence of the Fld/FldR/NADPH reducing
system and a 13ꢀaa peptide corresponding to residues 83ꢀ95
of the Bt S12 protein, formation of SAH (m/z = 385.0), 5'ꢀ
dAH (m/z = 252.1) and methylthiolated peptide, 3-MS-1
(m/z = 760.1) was observed by LC/MS (Figure 1). Interestꢀ
ingly, however, the final concentrations of SAH and 5’ꢀdAH
were 1.1ꢀ to 2.5ꢀfold higher than that of the methylthiolated
product, which is similar to that observed when assays were
conducted using dithionite as the requisite source of elecꢀ
trons. To determine initial rates of formation for SAH, 5’ꢀ
dAH and 3-MS-1, the amplitudes of each of the correspondꢀ
ing curves were multiplied by the firstꢀorder rate constants
obtained from fits of the data to a singleꢀexponential equaꢀ
tion, resulting in rates of 19.7 ± 0.68 ꢁM minꢀ1 (SAH) 6.03 +
0.01 ꢁM minꢀ1 (5’ꢀdAH), and 3.95 + 0.01 ꢁM minꢀ1 (3ꢀMSꢀ1).
These results suggest that methylation of RimO by SAM
takes place relatively rapidly as compared to formation of 5’ꢀ
dAH and product and that the Ec Fld/FldR/NADPH reducꢀ
ing system is indeed capable of delivering electrons to Bt
RimO for catalysis. Interestingly, while formation of 5’ꢀ
dAH—even in the presence of the in vivo reducing system—
is in excess of methylthiolated product, reactions conducted
in ~90% D2O result in no 5’ꢀdAD above natural abundance.
This observation suggests that any 5’ꢀdA• that does not lead
to the correct product abstracts an enzymeꢀ or substrateꢀ
derived Hꢀatom that is not from a solvent exchangeable site
(SI Figure 7).
Figure 1. Bt RimOꢀcatalyzed timeꢀdependent formation of
SAH, 5'ꢀdAH, and methylthiolated product (3-MS-1) with
the Ec Fld/FldR/NADPH reducing system. The reaction was
conducted as described in the supporting information and
contained 100 ꢁM Bt RimO, 3 mM SAM, 3 mM peptide (1),
200 ꢁM Fld, 50 ꢁM FldR, 3 mM NADPH, and 50 mM Naꢀ
HEPES pH 7.5.
To determine the stereoselectivity of Hꢀatom abstracꢀ
tion, deuteriumꢀcontaining isotopomers (pro-R or pro-S) at
C3 of aspartate were incorporated into peptide substrates,
which were subsequently used in the RimO reaction. The
synthesis of the 3ꢀpro-R and 3ꢀpro-S deuterated substrates
followed the approaches described by Young et al.22 and
Richards et al. (SI Scheme 1),23 which exploits the ability
of aspartate ammoniaꢀlyase (AAL) to catalyze the stereoseꢀ
lective incorporation of deuterium from D2O into the C3
pro-R position of aspartate when incubated with fumarate
and excess ammonium chloride to afford (2S,3R)ꢀ3ꢀ[2H1]
aspartate. Similarly, AAL catalyzes the stereoselective inꢀ
corporation of a proton in the C3 pro-R position of asparꢀ
tate, and when incubated with [2,3ꢀ2H2]ꢀfumarate and amꢀ
monium chloride in H2O, affords (2S,3S)ꢀ[2,3ꢀ2H2] asparꢀ
tate.23 We confirmed the selective deuterium incorporation
1
in both labeled aspartates by H NMR (SI Figures 2 and 3)
after converting their side chain carboxylic acids to tertꢀ
butyl esters and protecting their amine groups with Fmoc.
Displayed in panels A, B, and C of Figure 2 are the expandꢀ
1
ed regions (2.4 to 4.7 ppm) of the H NMR spectra of the
unlabeled, pro-R labeled, and pro-S labeled protected asꢀ
partates, respectively, where the C2 and C3 proton signals
are observed. The C3 hydrogens of unlabeled FmocꢀAspꢀβꢀ
OtBu in panel A exhibit both geminal coupling and vicinal
coupling to the hydrogen on C2, resulting in two sets of
doublets of doublets. The doublet of doublets correspondꢀ
ing to the pro-R hydrogen is centered at 2.77 ppm, while
that of the pro-S hydrogen is observed at 2.98 ppm. In panꢀ
el B, the replacement of the pro-R hydrogen with deuterium
results in the disappearance of the doublet of doublets at
2.83 ppm. Additionally, one of the doublets at 2.98 ppm
that was present due to geminal coupling is now absent,
confirming that the pro-R deuterium was indeed retained.
Similarly, in panel C, the replacement of the pro-S hydrogen
at C3 and the hydrogen at C2 with deuterium eliminated
geminal and vicinal proton coupling, and resulted in the
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collapse of the doublet to a single peak at 2.77 ppm, correꢀ
centrations of methionine and 5'ꢀdAH formed in a reaction
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sponding to the C3 pro-R hydrogen. The absence both of
the proton signal at 2.98 ppm and of proton coupling conꢀ
firmed the presence of deuterium at C2 and in the pro-S
position at C3.
of Bt RimO incubated under turnover conditions with 1 and
the Ec Fld/FldR/NADPH reducing system were quantified
and compared (SI Figure 6). Methionine and 5'ꢀdAH were
formed in a 1:1 ratio, thereby ruling out methionine as a
source of sulfide/methylthio group for the RimO reaction.
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Figure 2. H NMR spectra of unlabeled (A), pro-R labeled
(B) and pro-S labeled (C) aspartate with its amino and βꢀ
carboxylic acid moieties protected with Fmoc and tertꢀbutyl
ester groups, respectively.
Catalysis by RimO is believed to involve Hꢀatom abꢀ
straction from C3 of aspartate by a 5’ꢀdA• generated from
the reductive cleavage of SAM. To determine the overall
stereochemical course of the RimO reaction, the stereoselecꢀ
tively labeled aspartates were appropriately protected for
solid phase peptide synthesis and incorporated at the target
position of the 13ꢀmer peptide derived from the Bt S12 proꢀ
tein. The peptide containing the C3 pro-R aspartate (2) and
the peptide containing the C3 pro-S aspartate (3) were used
as substrates in reactions with Bt RimO to determine which
of the two peptides supports the formation of 5'ꢀ
deoxyadenosine enriched with deuterium (5'ꢀdAD), thereby
indicating which of the Hꢀatoms attached to C3 is abstractꢀ
ed. Panels A, B, and C of Figure 3 show the timeꢀ
dependent formation of 5'ꢀdAH, 5'ꢀdAD, and methylthiolꢀ
containing product (3ꢀMS) obtained using peptides 1, 2, or
3, respectively. Formation of 5’ꢀdAD is only observed with
peptide 3 containing the C3 pro-Sꢀlabeled aspartate, indiꢀ
cating that the 5’ꢀdA• abstracts the pro-S Hꢀatom from the
substrate and that the overall RimO reaction proceeds with
inversion of configuration. A primary kinetic isotope effect
of ~1.9 was calculated from the ratios of the rates of 5’ꢀdAH
formation with peptide 1 (5.41 ± 0.01 ꢁM minꢀ1) and 5’ꢀdAD
+ 5’ꢀdAH formation with peptide 3 (2.88 + 0.01 ꢁM minꢀ1),
indicating that Hꢀatom abstraction is at least partially rateꢀ
limiting. Furthermore, there appears to be a substantial
secondary isotope effect (~1.4) in the reaction using the 3ꢀ
pro-Rꢀlabeled substrate (2), given the rates of 5’ꢀdAH forꢀ
mation with peptide 1 (5.41 ± 0.01 ꢁM minꢀ1) versus peptide
2 (3.7 ± 0.01 ꢁM minꢀ1).
Figure 3. Bt RimO catalyzed reactions at 37 ˚C in the presꢀ
ence of Ec Fld/FldR/NADPH, SAM and peptides 1 (A), 2
(B), or 3 (C). The reactions were conducted as described in
the supporting information and contained 100 ꢁM Bt RimO,
3 mM SAM, 3 mM peptide (1, 2, or 3 as indicated), 200 ꢁM
Fld, 50 ꢁM FldR, 3 mM NADPH, and 50 mM NaꢀHEPES pH
7.5.
Previous studies of Tm RimO conducted by two differꢀ
ent laboratories shed light on the mechanism by which this
enzyme catalyzes methylthiolation of D89.6,7,18,19 Contrary
to the initial proposed mechanism, based on the mechaꢀ
nisms of the RS thiotransferases BioB15,24ꢀ29 and LipA,11,30 in
which the auxiliary clusters of these enzymes were shown to
be the sacrificial source of sulfide, RimO has been shown to
synthesize an ꢀSCH3 group that is presumably bound to the
unique Fe ion of its auxiliary cluster.18,19 Subsequent generaꢀ
tion of the 5'ꢀdA• for Hꢀatom abstraction, now known from
this study to be the pro-S Hꢀatom, generates a substrateꢀ
based radical with which the synthesized ꢀSCH3 group comꢀ
bines to form the methylthiolated product. Although the
details of the attachment of the methylthio group onto the
substrate remain elusive, the determination of both the steꢀ
reospecificity of Hꢀatom abstraction and the absolute conꢀ
figuration at C3 of 3ꢀMSꢀD89 allows us to conclude that ꢀ
SCH3 insertion occurs with inversion of configuration. Toꢀ
gether, these results make RimO the third RS enzyme for
which the stereochemical outcomes of Hꢀatom abstraction
and sulfur/methylthio group insertion is known.12,17
The finding that the Ec Fld/FldR reducing system acts
as a competent source of reducing equivalents for the Bt
RimO reaction led us to believe that it would allow demonꢀ
stration of the expected product ratios of 1:1:1 (methylthioꢀ
lated product:SAH:5’ꢀdAH). Surprisingly, 5'ꢀdAH and SAH
formation in excess of product was still observed, mirroring
the results that we, and others, have reported for Tm
RimO.7,18,19 In previous studies of the Tm RimO reaction, in
which the chemical reducing agent, sodium dithionite, was
used, formation of both SAH and 5'ꢀdAH in 2ꢀfold or greater
The source of sulfur that is inserted in the RimO meꢀ
thylthiolation reaction has yet to be definitively determined;
however, it has been proposed that the auxiliary Nꢀterminal
[4Feꢀ4S] cluster serves this role,6 or serves as a binding site
for a sulfide species that is methylated and subsequently
inserted into the substrate in a radicalꢀdependent proꢀ
cess.18,19 To determine whether methionine—a byproduct
formed during the reductive cleavage of SAM—is used by
RimO as a source of sulfur in the methylthio group, the conꢀ
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excess of product was observed, which was attributed to
(9) Cicchillo, R. M.; Lee, K.ꢀH.; BaleanuꢀGogonea, C.;
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abortive cleavage of SAM, a known side reaction of RS enꢀ
zymes7,18,19. The use of the Ec Fld/FldR reducing system did
decrease the amount of SAH and 5'ꢀdAH formed per meꢀ
thylthiolated product; however, abortive cleavage still reꢀ
sulted. Reactions conducted in D2O demonstrated that solꢀ
vent and solvent exchangeable Hꢀatoms do not quench any
5'ꢀdA• formed productively or abortively, suggesting that
abstraction of an Hꢀatom derived from the RimO polypepꢀ
tide or the peptide substrate occurred. Future studies will
address the questions concerning the overall stoichiometry
of reactants and products and the source of sulfide in the
methylthiolation reaction.
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ASSOCIATED CONTENT
Supporting Information
Experimental methods, synthetic details, and additional
spectra and figures are provided. This material is available
free of charge via the internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
squire@psu.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank Dr. WeiꢀChen Chang for useful synthetic advice
and Dr. Carlos C. Pacheco for help with obtaining and anaꢀ
lyzing the NMR data. The National Institutes of Health
(NIH) (GM103268 to S.J.B.) provided funding for this reꢀ
search. NMR spectra were analyzed using MNova software
from MestreLab Research.
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