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org/biochemistry Article

New Role for Radical SAM Enzymes in the Biosynthesis of

Thio(seleno)oxazole RiPP Natural Products
Julia K. Lewis, Andrew S. Jochimsen, Sarah J. Lefave, Anthony P. Young, William M. Kincannon,
Andrew G. Roberts, Matthew T. Kieber-Emmons, and Vahe Bandarian*
Cite This: Biochemistry 2021, 60, 3347−3361 Read Online

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ABSTRACT: Ribosomally synthesized post-translationally modi-

Downloaded via UNIV ESTADUAL PAULISTA on January 22, 2022 at 18:40:04 (UTC).

fied peptides (RiPPs) are ubiquitous and represent a structurally

diverse class of natural products. The ribosomally encoded
precursor polypeptides are often extensively modified post-
translationally by enzymes that are encoded by coclustered
genes. Radical S-adenosyl-L-methionine (SAM) enzymes catalyze
numerous chemically challenging transformations. In RiPP biosynthetic pathways, these transformations include the formation of
C−H, C−C, C−S, and C−O linkages. In this paper, we show that the Geobacter lovleyi sbtM gene encodes a radical SAM protein,
SbtM, which catalyzes the cyclization of a Cys/SeCys residue in a minimal peptide substrate. Biochemical studies of this
transformation support a mechanism involving H-atom abstraction at the C-3 of the substrate Cys to initiate the chemistry. Several
possible cyclization products were considered. The collective biochemical, spectroscopic, mass spectral, and computational
observations point to a thiooxazole as the product of the SbtM-catalyzed modification. To our knowledge, this is the first example of
a radical SAM enzyme that catalyzes a transformation involving a SeCys-containing peptide and represents a new paradigm for
formation of oxazole-containing RiPP natural products.

R ibosomally synthesized post-translationally modified

peptides (RiPPs) are a structurally diverse class of natural
products. These peptides are synthesized on the ribosome and
are subsequently post-translationally modified to build in
functional and biological diversity.1,2 Over the last dozen years,
radical S-adenosyl- L -methionine (SAM) enzymes have
emerged as being involved in many chemically challenging
reactions in pathways that produce RiPP natural products.3
Members of the radical SAM superfamily are generally
identified on the basis of a conserved CX3CX2C motif,
whose thiolates are responsible for the coordination of the
three iron corners of a catalytic [4Fe−4S] metallocluster. The
fourth iron of this cluster coordinates the α-amino and α-
carboxylate moieties of SAM, binding the cofactor to the active
site.4−6 Radical SAM enzymes require a reductant, such as
dithionite (dT), to reduce the [4Fe−4S] cluster from a
catalytically inactive +2 state to a catalytically active +1 state.
The reduced [4Fe−4S] cluster cleaves SAM, generating a 5′-
deoxyadenosyl radical (dAdo•), which initiates the trans-
formation of the substrate, often by H-atom abstraction7,8
(Figure 1A). Although SAM is sometimes reformed during the
catalytic cycle by some, in many radical SAM enzymes, it is
used stoichiometrically.9
Many radical SAM proteins that function as RiPP maturases
install chemically distinct crosslinks in their respective natural
products.2 The most common of these reactions are thioether
crosslinks of varied regiochemistry. The first thioether
crosslinking enzyme to be identified, AlbA, is a sactipeptide
(sulfur to α-carbon thioether) synthase that catalyzes the
© 2021 The Authors. Published by
American Chemical Society
3347
insertion of three thioether crosslinks in the precursor peptide
of the antibiotic, subtilosin A10 (Figure 1B). More recently,
RiPP maturase proteins have been identified, which instead of
forming a C−S bond between a cysteine residue and a Cα
catalyzes a crosslink to either the Cβ11 or Cγ12 positions of the
acceptor amino acid, thereby expanding the regiochemical
outcomes.
The roles of radical SAM maturases in RiPP biosynthetic
pathways, however, are not limited to the formation of C−S
bonds. For example, PqqE catalyzes the formation of a C−C
bond between the Cγ of a glutamate and the C3 of a tyrosine
in the precursor to the bacterial redox cofactor pyrroloquino-
line quinone (PQQ), PqqA.13 The number of C−C bond
forming RiPP maturases has a large repertoire,14 including
StrB, an enzyme that catalyzes a crosslink between a lysine and
tryptophan residue in the biogenesis of streptide, a peptide
involved in quorum sensing15 (Figure 1B). Moreover, the
recent discovery that TqqB catalyzes the formation of an ether
crosslink between a glutamine and threonine residue has
broadened the scope of radical-mediated chemistry in RiPPs to
also include C−O bond formation.16 Additional examples of
Received: July 7, 2021
Revised: October 15, 2021
Published: November 3, 2021
https://doi.org/10.1021/acs.biochem.1c00469
Biochemistry 2021, 60, 3347−3361
Biochemistry pubs.acs.org/biochemistry Article

Figure 1. Radical SAM enzymes and RiPP maturation. (A) Reductive cleavage of SAM to generate a 5′-deoxyadenosyl radical (dAdo•) is catalyzed
by a [4Fe−4S] cluster. The sulfur and iron ions are denoted in yellow and red, respectively. The dAdo• initiates the catalytic cycle and either the
cofactor is reformed at the end of the cycle or used it stoichiometrically. (B) Examples of RiPP natural products with C−S, C−C, and C−O bonds
are installed by radical SAM enzymes. (C) Gene cluster from Geobacter lovleyi contains a gene coding for a radical SAM enzyme (sbtM), which is
coclustered with one coding for a precursor peptide (sbtMa).20 The DNA sequences of the SbtMa peptide contain one or more SeCys (U) coding
amber codons. The minimal sequence used in this study is shown with U or C at the site modified by SbtM shown in red.

radical-mediated transformations in RiPP pathways also
include protein splicing to remove tyramine within a precursor
protein to form an α-keto-β-amino amide,17 with epimerization
occurring at the α-carbon of various amino acids.18,19
Therefore, radical SAM RiPP maturases catalyze the formation
of C−H, C−C, C−O, and C−S bonds in their respective
substrates (Figure 1B).
The radical SAM peptide-maturase genes are often co-
clustered with an open reading frame (orf) encoding the
putative peptide substrate, as well as those coding for any other
tailoring proteins that are required to synthesize the full natural
product. Several years ago, Haft and Basu surveyed bacterial
genomes and identified potential RiPP gene clusters that
encode at least one radical SAM enzyme and a putative peptide
substrate20 (Figure 1C).
One of these clusters, initially called the SCIFF proteins (six
cysteines in 45), has since been shown to catalyze a C−S
crosslink between a Cys thiol and a carbon of a Thr residue.21
The SCIFF family is part of a larger group of ranthipeptides
that catalyze thioether crosslinks to positions other than the α
carbon of an amino acid.2
One of the clusters identified in this study was predicted to
encode a selenocysteine (SeCys)-containing peptide natural
product with the necessary selenocysteine insertion elements
(SECIS) also present. As with other bacterial SeCys-containing
proteins, SECIS elements are comprised of an amber UGA
stop codon, with a predicted stem-loop structure located
immediately downstream of the peptide orf.22,23 SeCys is a rare
amino acid, and to our knowledge, there are no known
examples of post-translationally modified SeCys-containing
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RiPP natural products. On the basis of sequence alignment, the
proximal maturase (sbtM) is predicted to be a member of a
subgroup of radical SAM enzymes, which has a cysteine-rich
C-terminal extension that coordinates two auxiliary [4Fe−4S]
clusters (Figure S1). These additions to the radical SAM core
are termed SPASM domains, so-called because they were
initially identified in the biosynthesis of subtilosin, PQQ,
anaerobic sulfatase, and mycofactocin biosynthesis. While
SPASM domains are prevalent in radical SAM enzymes that
modify peptides, the exact function of these auxiliary clusters
remains unknown. However, where available, data suggests
that they are required for overall activity and not for the
cleavage of SAM.21,24−30
The study that identified the putative SeCys RiPP cluster
postulated that these peptides could be of different lengths,
some with potentially two SeCys modification sites.20 In
general, these putative peptides have a relatively well-conserved
leader sequence, which is followed by a core region that
contains the SeCys residue(s). Unlike the N-terminus,
however, C-terminal regions are hypervariable in sequence.
While this study clearly identified a group of novel peptide-
maturase pairs, it did not provide any insight as to the nature of
the modification(s) that are installed by the radical SAM
enzyme. In the intervening time since the paper20 was
published, NCBI now identifies homologs of this radical
SAM maturase as SbtM and the peptide a selenobacterocin.
Interestingly, some of the peptides identified in the original
study have since been updated to indicate the presence of at
least two SeCys residues. To our knowledge, these peptides
have not been detected in nature, and so the identity of the
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modification, the length of the peptide, or the SeCys content
are all unknown. The sequence similarity network (SSN) of
SbtM (Figure 2) shows that it coclusters in the network with
Figure 2. SSN of peptide modifying radical SAM enzymes. The amino
acid sequences of several characterized SPASM domain-containing
proteins were individually subjected to BLAST analyses, and the
resulting sequences were analyzed by the suite of programs at EFI
(https://efi.igb.illinois.edu). Detailed procedures are in the Support-
ing Information.31
other proteins of unknown function, which are often annotated
as selenobiotic biosynthesis enzymes. Members of Geobacter
and Desulfobacteria make up most of the closest neighbors in
the cluster.
In this paper, we characterize the reaction between the
radical SAM enzyme, SbtM, and a 53 amino acid peptide
substrate reported by Haft and Basu.20 The peptide contains
the first SeCys site, as well as additional amino acids beyond
the SeCys that are conserved in other members of the family,
but dispenses with the hypervariable region. Our data show
that the radical SAM enzyme is a new member of the RiPP
family that introduces a thiooxazole moiety in the peptide-
containing Cys. The modification also occurs when SbtM is
incubated with the SeCys-containing peptide, which is
presumably the biologically encoded amino acid. Site-directed
mutagenesis studies show that while SeCys and Cys are
tolerated at the position, Ala and Ser are not transformed. The
cyclization is accomplished by the removal of two hydrogen
atoms from the β-carbon of Cys, one proton from the Cys
amide nitrogen, and a fourth proton from the α-carbon of the
peptide. The radical SAM enzyme is introducing a C−O
linkage to form an oxazole functional group in the peptide,
expanding the repertoire of modifications installed by radical
SAM RiPP maturases.
■ EXPERIMENTAL PROCEDURES
Cloning and Expression of SbtM. The sbtM gene
(WP_083768621) was codon-optimized and cloned into the
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pET28a(+)-TEV plasmid by Genscript using the restriction
sites of NdeI and XhoI. The codon-optimized gene sequence of
sbtM from G. lovleyi is shown in Figure S2, and the protein
sequence is shown in Figure S3.
The plasmids, sbtM-pET28a(+)-TEV and pDB1282, were
cotransformed into Escherichia coli BL21 (DE3) T1 resistant
cells (NEB C2527). The plasmid pDB1282 contains the isc
operon from Azotobacter vinelandii under arabinose control.
The operon is often included in the expression of radical SAM
enzymes because it assists in the assembly of iron−sulfur
clusters in heterologously expressed proteins.32,33 The trans-
formation mixture was plated onto plates containing Lennox
broth (LB) agar supplemented with 34 μg/mL kanamycin, 100
μg/mL ampicillin, and placed at 37 °C overnight. A single
colony was selected from the plate and used to inoculate an
overnight culture (0.1 L) of LB containing 34 μg/mL
kanamycin and 100 μg/mL ampicillin grown at 37 °C with
shaking at 175 rpm. An aliquot (0.01 L) of the overnight
culture was used to inoculate six 2.8 L Fernbach flasks
containing 1 L each of LB supplemented with 34 μg/mL
kanamycin and 100 μg/mL ampicillin. The cultures were
grown at 37 °C and 175 rpm to an OD600nm of ∼0.5. At this
point, the cultures were supplemented with 0.05 mM iron(III)
chloride hexahydrate and 0.05 mM cysteine, and expression of
the isc operon from the pDB1282 was induced with the
addition of 0.05% (w/v) arabinose. The temperature and
shaking were then reduced to 18 °C and 120 rpm, respectively.
At an OD600nm ∼ 0.7, expression from the sbtM-pET28a(+)-
TEV plasmid was induced with the addition of 0.1 mM
isopropyl β-D-1-thiogalactopyanoside (IPTG). The cultures
were grown overnight (∼18 h), and the cells were harvested by
centrifugation at 5000g. The cell pastes were flash-frozen in
liquid nitrogen and stored at −80 °C until use.
Purification of SbtM. The purification of SbtM was
performed in a Coy Laboratories anaerobic chamber
maintained with 97% N2/3% H2 atmosphere. The cell paste
(20 g) was placed in a metal beaker and resuspended in ∼0.2 L
of 0.05 M KPi (pH 7.4) buffer containing 0.5 M KCl, 0.05 M
imidazole, 100 μg/mL lysozyme, and 1 mM phenylmethane-
sulfonyl fluoride (PMSF). The cells were lysed by a Branson
digital sonifier operated at 50% amplitude for a total of 12 min
(15 s on/45 s off) while stirring on ice. The suspension was
centrifuged at 18 000g for 35 min at 4 °C. The clarified lysate
was then loaded onto a 5 mL of nickel sulfate-charged HisTrap
HP column (GE Healthcare) that had been equilibrated with
loading buffer containing 0.05 M KPi (pH 7.4), 0.5 M KCl,
and 0.05 M imidazole. The column was washed with five
column volumes (CV) of loading buffer, and the enzyme was
eluted with a linear gradient over 8 CV to 0.5 M imidazole in
the loading buffer. All fractions containing SbtM were
identified via color and/or sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE) gel. The pooled
fractions were desalted on a BioGel P6 DG desalting gel 100−
200 mesh (wet) (Bio-Rad) into 0.02 M Tris·HCl (pH 8.0)
containing 5 mM dithiothreitol (DTT).
The resulting protein was further purified by anion exchange
chromatography as follows. The desalted fractions from the
affinity step were loaded onto a Q-Sepharose FF column (11
cm × 3 cm) (GE Healthcare), which had been equilibrated
with buffer containing 0.02 M Tris·HCl (pH 8.0) and 5 mM
DTT. SbtM was eluted with a 0.2 L linear gradient to 0.5 M
KCl in the loading buffer. Fractions containing the desired
protein were identified by color and/or SDS-PAGE gel and
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desalted into buffer containing 0.05 M PIPES·NaOH (pH 7.4),
0.15 M KCl, and 5 mM DTT. The protein was then flash-
frozen with liquid nitrogen and stored at −80 °C.
The concentration of the desalted SbtM was determined by
the Bradford method, using bovine serum albumin (BSA) as a
standard. The protein was reconstituted by mixing with 15−25
molar equiv. of FeCl3 hexahydrate (dissolved in saturated
sodium bicarbonate) and Na2S nonahydrate (in water). The
iron(III) chloride hexahydrate was added dropwise to the
protein followed by sodium sulfide, which was dissolved in
water. The reconstitution mixture was stirred for 4 h at room
temperature. The resulting mixture was clarified by centrifu-
gation at 16 000g for 5 min to remove any debris and desalted
on a BioGel P6 DG desalting gel 100−200 mesh (wet) (Bio-
Rad) into buffer containing 0.05 M PIPES·NaOH (pH 7.4),
0.15 M KCl, and 5 mM DTT. The protein was concentrated to
∼1 mL with an Amicon concentrator under N2 with a YM-10
membrane (Millipore).
The reconstituted enzyme was further purified by a
Sephacryl 16/60 S-200 column equilibrated with buffer
containing 0.05 M PIPES·NaOH (pH 7.4), 0.15 M KCl, and
5 mM DTT. The protein was eluted isocratically at 1 mL/min,
and fractions containing SbtM were identified by color and/or
SDS-PAGE gel. The pooled fractions were concentrated to
∼0.5 mL, aliquots were flash-frozen in liquid nitrogen, and
stored at −80 °C. SbtM was quantified by a Bradford assay
with BSA as a standard.
Amino Acid Analysis and Iron Concentration Deter-
mination. To obtain a correction factor to correlate the
Bradford assays to the actual protein concentration, five
independent preparations of SbtM were subjected to amino
acid analysis (by the Molecular Structure Facility at the
University of California-Davis). For the analysis, the protein
samples were hydrolyzed under vacuum in a solution
containing 6 N HCl and 1% phenol at 110 °C. The samples
were resuspended with 40 nmol/mL norleucine as an internal
standard. The samples were analyzed by a Hitachi 8800
analyzer that had been calibrated with amino acid standards for
protein hydrolysate on the Na-based Hitachi 8800 (Sigma, A-
9906). These standards were verified by the National Institute
of Standards and Technology (NIST) standard reference
material 2389a. The protein samples were injected onto a
Concise ion-exchange column (AminoSep Beckman Style Na+,
part #AAA-99-6312) with a secondary reaction with ninhydrin
for detection that uses Pickering Na buffers. The correction
factor for the Bradford assays was thus determined to be 0.61.
The iron content of the reconstituted protein was determined
through inductively coupled plasma-mass spectrometer (ICP-
MS) on two separate enzyme preparations at the Center for
Water, Ecosystems, and Climate Science in the Department of
Geology and Geophysics at the University of Utah. For the
analysis, enzyme preparations were diluted to a concentration
of 2−10 μM trace metal grade nitric acid prior to submission.
The determination of iron in soluble protein solutions was
performed with a triple quadruple inductively coupled plasma-
mass spectrometer (ICP-MS, Agilent 8900, Santa Clara, CA)
at the ICP-MS Laboratories, Dept. of Geology and Geophysics,
University of Utah. Protein solutions were diluted 1:40 with
2.4% HNO3 and 10 ng In/mL was added as internal standard.
An external calibration curve was prepared from 1000 mg/L
single elemental standard (Inorganic Ventures, Christiansburg,
VA). Concentrations of Fe in the six calibration solutions were
0, 8.3, 20.7, 66.2, 165.5, and 331.1 ng Fe/mL, respectively, and
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all contained 10 ng In/mL. Diluted samples, blanks, and the
calibration solutions were run in the ICP-MS using a double-
pass quartz spray chamber, PTFE nebulizer and dual-syringe
introduction system (Teledyne, AVX71000), platinum cones,
and sapphire injector in a quartz platinum-shielded torch. Fe
and In were detected at masses of 56 and 115, respectively,
with a flow of 8 mL He/min in the collision cell. Limit of
determination (LoD) was calculated as 3 times the standard
deviation of the blanks, multiplied by the 40-fold dilution
factor used for samples. The instrument is located in a filtered
air positive pressure lab, and sample handling and dilutions
were performed in laminar flow benches and using calibrated
pipettors (Eppendorf Reference, Hamburg, Germany). Certi-
fied Reference Material CRM 1643f (National Institute of
Standards and Technology, Gaithersburg, MD) was diluted
1:20 and run together with the samples and external calibration
curve as quality control for the calibration. The measured value
for Fe in CRM 1643f agreed with the certified value within
10%.
Synthesis of Unlabeled, Cys35SeCys, [13C2,15N]-Gly34,
[ C3,15N]-Cys35, [2,2 D2]-Gly34, and [3,3 D2]-Cys35
13
SbtMa Peptides. All SbtMa peptides and variants were
synthesized on a PS3 peptide synthesizer (Protein Technol-
ogies Inc.) at a 0.025 mmol scale using solid-phase peptide
synthesis (SPPS) methodology. The isotopically enriched
Fmoc-[13C2,15N]-Gly, Fmoc-[13C3,15N]-Cys S-trityl, Fmoc-[2,2
D2]-Gly, and Fmoc-[3,3 D2]-Cys S-trityl were purchased from
Cambridge Isotope Laboratories. The Fmoc-SeCys(Mob)-OH
was purchased from Sigma-Aldrich. The syntheses were carried
out essentially as described by Bruender et al.,21 with the
following modifications. After precipitating the peptide in ice-
cold diethyl ether, the solution was poured over a Büchner
funnel and vacuumed to collect the peptide and continued to
dry for ∼1 h prior to being resuspended in ∼40 mL of water.
The suspension was then placed in a sonicator bath for 30 min
to aid in the dissolution of the peptide, flash-frozen in liquid
nitrogen, and lyophilized. The cleavage and deprotection of
SbtMa peptide-containing SeCys at position 35 followed the
procedure described by SteMarie et al.,34 except that the
cleavage buffer contained 2% 2,2′-dithiobis-5-nitropyridine
(DTNP) and did not include thioanisole. The resulting Sec-5-
Npys adduct was deprotected with 5 equiv of ascorbate (pH
4.5) at 25 °C for 4 h and stirred for 3 h in 10 mM DTT to
reduce diselenides and high-performance liquid chromatog-
raphy (HPLC)-purified on a Phenomenex Jupiter C12
preparative column (21.2 mm × 250 mm, 4 μm particle size,
90 Å pore size) with buffer A as 0.1% trifluoroacetic acid
(TFA, HPLC Grade) in nanopure water and buffer B as 0.1%
TFA (HPLC Grade) in acetonitrile (HPLC Grade). The
separation was run at 5 mL/min with a linear gradient of buffer
A from 88 to 60% for 95 min. The column was washed with
100% buffer B from 96 to 125 min and returned to 88% buffer
A from 125 to 130 min, followed by and reequilibration with
88% buffer A from 130 to 155 min. Fractions containing the
desired peptide were identified via LC-MS using one of two
instrumental setups (Ultimate 3000 HPLC with a diode-array
detector interfaced to a LTQ OrbiTrap XL mass spectrometer
or a Vanquish UHPLC with a diode-array detector connected
to a Q-Exactive mass spectrometer) fitted with a Hypersil
GOLD C4 column (4.6 mm × 250 mm, 5 μm particle size) for
separation at 0.2 mL/min. For the LC-MS program, buffer A
was LC-MS Optima water (Fisher)/0.1% (v/v) LC-MS
Optima TFA (Fisher) and buffer B was LC-MS Optima
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acetonitrile (Fisher)/ 0.1% (v/v) LC-MS Optima TFA
(Fisher). The 15 min separation consisted of washing the
column with 100% A for 3 min, followed by a linear gradient to
100% B from 3 to 6 min, followed by washing with 100% B
from 6 to 8 min. The column was returned to 100% A over 3
min and equilibrated in the starting conditions for an
additional 3 min. The MS detectors were operated in positive
ion mode, and FT analyzer settings are as follows: 100 000
resolution for the LTQ OrbiTrap and 70 000 resolution for the
Q-Exactive, 1 microscan, and 200 ms maximum injections
time. All data were analyzed on Xcalibur software (Thermo
Fisher).
Enzymatic Reaction of SbtMa with SbtM. All
modification reactions were conducted at room temperature
in a Coy Laboratories anaerobic chamber with 97% N2/3% H2
atmosphere. The reactions contained 0.05 M PIPES·NaOH
(pH 7.4), 10 mM DTT, 10 mM dT, 2 mM SAM
(enzymatically synthesized and purified as previously
described35,36), ∼0.8 mg/mL SbtMa, and 0.015−0.03 mM
SbtM, in a total volume of 0.1 mL. Control reactions were also
conducted in the absence of dT, SAM, and SbtM, respectively.
The reactions were initiated with the addition of the peptide
substrate and quenched after at least 4 h with the addition of
30 μL of 30% (w/v) trichloroacetic acid (TCA, ACS grade).
The samples were centrifuged at 10 000 rpm in a micro-
centrifuge to remove the precipitated enzyme.
Alkylation of SbtMa. Where alkylation by iodoacetamide
was desired, Tris·HCl (pH 8.0) was added to a final
concentration of 0.1 M to the quenched peptide reactions
followed by iodoacetamide to a final concentration of 30 mM.
The mixtures were incubated for 1 h in the dark, and the excess
iodoacetamide was quenched by the addition of DTT to 30
mM final concentration.
U/HPLC-MS Analysis of Enzymatic Reactions. The
enzymatic reactions were analyzed using either an Ultimate
3000 HPLC with a diode-array detector connected to a LTQ
OrbiTrap XL mass spectrometer or a Vanquish UHPLC with a
diode-array detector connected to a Q-Exactive mass
spectrometer. The LTQ OrbiTrap XL or Q-Exactive was
operated in positive ion mode, and the FT analyzer was set to
100 000 resolution, 1 microscan, and 200 ms maximum
injection time. The data were analyzed using Xcalibur software.
For the analysis, an aliquot (98 μL) was injected onto a
Hypersil GOLD C4 column (4.6 mm × 250 mm, 5 μm particle
size) (Thermo Fisher) pre-equilibrated in 0.1% (v/v) LC-MS
Optima TFA (Fisher) in LC-MS Optima water (Fisher). The
separation was carried out at 0.2 mL/min with buffer A 0.1%
(v/v) TFA in water and buffer B Optima grade acetonitrile
with 0.1% (v/v) TFA. The separation utilized the following
program: washing with 0% B from 0 to 3 min; linear gradient
to 99% B from 3 to 28 min; washing with 100% B from 28 to
34.9 min; and equilibration at 0% B from 35 to 40 min.
Collision-Induced Dissociation (CID) Fragmentation
of Unmodified and Modified SbtMa. To obtain sufficient
material for fragmentation by direct infusion, the reactions
described above were scaled up 5-fold to 0.5 mL and the
SbtMa was increased to ∼2 mg/mL. Following TCA treatment
and centrifugation, the reaction mixtures were desalted by C4
ZipTips (Millipore) following the manufacturer’s protocols.
The analyzer was first tuned to the mass of the SbtMa. For the
analyses, the +4 charge state envelope corresponding to the
modified and unmodified SbtMa was isolated in the CID cell
using an isolation width of 1.0 m/z and 0.1 ms activation time
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and fragmented with a collision energy of 20%. The analysis of
fragmentation products used mMass software.
NMR of Unmodified and Modified SbtMa. The
modification reactions were scaled up to 1 mL and contained
0.05 M PIPES·NaOH (pH 7.4), 10 mM DTT, 10 mM DT, 2
mM SAM, ∼0.1 mg/mL SbtMa, and 7−13 μM SbtM. To
obtain sufficient material, ∼60 individual (1 mL) reactions
were combined after overnight incubation. The reactions were
pooled and lyophilized and resuspended in ∼5 mL of water,
filtered through a sterile syringe filter with a 0.2 μm
polyethersulfone membrane (VWR), and purified via a
Phenomenex Jupiter C12 prep column following the procedure
detailed above for SPPS peptides. The fractions containing
modified peptide were identified by LC-MS analysis of the
fractions, pooled, and lyophilized. These modified peptides
were used for the 13C experiments. The lyophilized modified
peptide was then resuspended in 50 mM Tris·HCl (pH 8.0)
and reacted with 20−30 mM iodoacetamide for 1 h in the
dark. The reaction was quenched by the addition of 30 mM
DTT and purified on the Phenomenex Jupiter C12 prep
column with the same procedure detailed for SPPS peptides.
These modified peptides were used for the 15N NMR
experiments. The unmodified SbtMa samples were prepared
by resuspending lyophilized SbtMa in 50 mM Tris·HCl (pH
8.0) and following the same iodoacetamide procedure and
purification as for the modified samples. The lyophilized
peptide was dissolved in 50 mM KPi (pH 6.0) 10% D2O for
15
N-HSQC and HNCACB experiments. For the 13C experi-
ments, lyophilized unmodified peptide was resuspended in 50
mM KPi (pH 6.0), 2 mM DTT, 100% D2O. The NMR spectra
were acquired on an Agilent DirectDrive 500 MHz NMR
spectrometer at the D.M. Grant NMR Center at the University
of Utah. The 2D-NMR data were analyzed by VNMRJ
software, and the 1D-NMR data and heteronuclear multiple
bond correlation (HMBC) were analyzed by Mestrenova
software.
FTIR of Unmodified and Modified SbtMa. Lyophilized
SbtMa peptide was mixed with dry KBr using an agate mortar
and pestle and pressed into a pellet. FTIR measurements were
run on a Bruker 80v interferometer with a DLaTGS detector, 4
cm−1 resolution, and 128 scans. Measurements with modified
or unmodified SbtMa were carried out under vacuum at 4.32
mbar and illuminated with a globar MIR source of non-
polarized light. Transmission spectra were processed using an
auto spline function implemented in the OPUS software.
Density Functional Theory (DFT) on Unmodified and
Modified Truncated SbtMa. Density functional theory
calculations were performed with Gaussian16 Version A.03.37
Truncated peptide models CH3−Gly−Cys−CH3 were built in
GaussView and truncated with a methyl group at the N− and
C− terminal sides of the Gly−Cys fragment. Both unmodified
and modified truncated peptides were optimized using the
B3LYP functional38−41 with the 6-311G basis set for all atoms.
Normal vibrational modes were extracted from analytical
frequency calculations and scaled by 0.966 due to the tendency
of the B3LYP to overestimate covalency.42
■
RESULTS
Protein Expression and Purification. The gene encoding
the putative radical SAM maturase, sbtM, was codon-optimized
and cloned into pET28a(+)-TEV plasmid for heterologous
expression in E. coli. To ensure that protein was replete with
requisite [4Fe−4S] cofactors, the cells were cotransformed
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Figure 3. SbtM modifies SbtMa. The panels show the +4 charge state of the mass spectra of the SbtMa-treated ± SAM, ± dT, or ± SbtM. The
orange and blue lines mark the positions of monoisotopic peaks for modified and unmodified peptides, respectively. The spectra in (A) correspond
to reactions containing Cys-SbtMa. The calculated m/z for unmodified and modified SbtMa occur at 1273.8976 and 1272.8898 amu, respectively,
and the measured monoisotopic features are within 3.1 ppm of the theoretical. In (B), the spectra corresponding to carbamidomethylated peptides
are shown. The calculated m/z for the monoisotopic peak before and after modification occurs at 1288.1530 and 1287.1452 amu, respectively. The
measured values are within 2.9 ppm of the theoretical. In (C), spectra obtained with SeCys-SbtMa after carbamidomethylated are shown. The
calculated m/z for the monoisotopic peak before and after modification occur at 1300.1391 and 1299.1313 amu, respectively. The measured values
are within 0.8 ppm of the theoretical. In (D), the UV−visible spectra of the modified Cys-SbtMa (blue), modified SeCys-SbtMa (pink), and
unmodified Cys-SbtMa (black) peptides are compared. The modified peptides exhibit a feature at 304 and 313 nm (λmax), respectively, suggesting
the presence of a conjugated unsaturation. The spectra of the SeCys and Cys-containing peptides shown in (D) are normalized to the λmax of the
respective peptide. The spectrum of the unlabeled peptide was normalized at 304 nm, whereas the peptide from a control reaction that did not
include SbtM does not. By contrast, the SeCys-containing peptide shows a red shift to 313 nm. This data support the formation of a conjugated
unsaturation in the peptide.

with pDB1282. This plasmid contains the isc operon from A.
vinelandii, which previous studies have shown aid in iron−
sulfur cluster assembly.32,33 The enzyme was purified via His6
affinity chromatography followed by a Q-Sepharose anion
exchange under anaerobic conditions. Sequence analysis
(Figure S1) suggests that protein is likely to have three
[4Fe−4S] clusters; therefore, the maturase was reconstituted
with 15−25-fold molar excess iron and sulfide. The
reconstitution step was followed by gel filtration, and the
purified protein is judged to be >95% pure based on SDS-
PAGE analysis (Figure S4). To permit accurate quantification
of cluster stoichiometry, amino acid analysis was carried out
3352
revealing a correction factor of 0.61 for Bradford assays with
BSA as standard. The preparations of protein reconstituted in
the presence of 25-fold excess iron contain 13.6 ± 1.9 mol,
whereas those reconstituted in the presence of lower
concentrations of iron were less replete. This is consistent
with the presence of at least two [4Fe−4S] clusters per
polypeptide chain. The putative minimal substrate SbtMa (see
Figure 1) was prepared by standard SPPS methods, as
described in the Experimental Procedures. The majority of
the experiments presented in this study utilize SbtMa-
containing Cys at position 35 because of its ease of synthesis
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and stability; however, a SeCys-containing analogue was also
prepared by SPPS and utilized as discussed below.
SbtM Modifies SbtMa. Preliminary studies demonstrate
that when SbtM is combined with Cys-containing SbtMa (see
Figure 1), a new product is formed in a manner that requires
the presence of SAM, dT, and the maturase (Figure 3).
Samples were analyzed by LC-MS employing high-resolution
mass analyzer, after quenching the reaction mixtures with TCA
(see Figure S5 for full spectra). The regions in the MS spectra
corresponding to the +4 charge state are shown in Figure 3A.
In the absence of the enzyme, the substrate displays a
monoisotopic peak at m/z of 1273.90, which is within 3.1 ppm
of the expected value for the unmodified peptide. However,
when SAM, dT, and SbtM are included, the envelope shifts the
monoisotopic peak to m/z of 1272.89. This overall mass
change of 4.03 amu is consistent with a modification that
potentially involves the loss of four protons.
To determine if the modification involved the thiol of the
Cys residue, reaction mixtures were treated with iodoaceta-
mide to alkylate the thiol prior to being analyzed by LC-MS.
The monoisotopic peak for the unmodified peptide under
these conditions is at m/z of 1288.16 (Figure 3B), which is
within 2.9 ppm of the expected value for a peptide containing a
single carbamidomethylated Cys residue. Interestingly, the
SbtM-modified peptide was alkylated as well, in addition to
exhibiting a monoisotopic mass shift of 4.03 amu relative to the
unmodified peptide. These observations suggest that the
modified peptide retains reactive sulfur, although the structure
of the modified moiety remains unknown.
To further probe the nature of the modification, we also
examined the UV−visible spectra of the peptide eluting from
the HPLC column in experiments, where a diode-array
detector was placed in-line with the MS instrument. Figure
3D compares the spectral data for modified and unmodified
peptides. To our surprise, the Cys-containing modified peptide
exhibits λmax = 304 nm.
We also synthesized the SbtMa peptide containing SeCys at
position 35 instead of Cys (see Figure 1), to determine if the
modification reaction occurs with SeCys, which is presumed to
be the naturally occurring amino acid. Due to the difficulty of
reducing diselenides reactions were subjected to alkylation by
iodoacetamide prior to analysis by LC-MS. Unlike the Cys-
containing peptides, Se gives rise to a complex pattern because
of the presence of numerous naturally occurring and abundant
stable isotopes (76Se, 9.23%; 77Se, 7.6%; 78Se, 23.69%, 80Se,
49.80; and 82Se, 8.82). In these experiments, the monoisotopic
peak of the +4 charge state of the peptide occurs at m/z of
1300.14, which is within 0.8 ppm of the expected value for a
singly carbamidomethylated SeCys residue (Figure 3C). The
observed +4 charge state envelope for the singly carbamido-
methylated peptide (Figure 3C) is identical to the simulated
envelope (Figure S6A). After incubation with dT, SAM, and
SbtM, however, the envelope shifts to place the monoisotopic
peak at m/z of 1299.13 (Figure 3C). The simulated envelope
of the +4 charge state for a peptide that has undergone a loss of
4.03 amu is consistent with the observed data (Figure S6B).
SbtM Catalyzes Modification of the GC Motif in
SbtMa. We next investigated the location of modification in
SbtMa via collision-induced dissociation (CID) of the
modified SbtMa by MS-MS fragmentation. The reaction
mixture and control reaction missing SbtM were introduced
into the mass spectrometer by direct infusion after quenching
with TCA and removal of salts. For the analysis, the envelope
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corresponding to the +4 charge state was isolated and
fragmented in the CID cell of the instrument. The
fragmentation data were analyzed by mMass, and all of the b
and y ions that were identified are listed in Tables S1 and S2
for the modified peptide and control peptide, respectively. The
y ions proved highly informative as a close examination of the
data reveals that both samples exhibit species with identical
mass up to y18. All species from y20 (+2 charge state) onwards
display a 2 amu difference between the modified and
unmodified samples, corresponding to a 4 amu difference.
The region in the fragmentation spectra that highlights this key
difference is shown in Figure 4A. These data show that the 4
amu loss observed in the presence of SbtM can be traced to
residues Gly34 and Cys35.
To probe the significance of this observation, peptides
containing Ala or Ser in the place of Cys35 were synthesized
and incubated with SbtM. Unlike the Cys- and SeCys-
containing counterparts, the Ala- and Ser-containing SbtMa
peptides exhibited identical mass spectral features after
incubation with SbtM, under conditions where parallel
experiments with the Cys-containing peptide showed a 4.03
amu loss (Figure S7). These observations are significant
because they suggest that while the modification maintains the
reactivity of the thiol/selenol residue, the identity of the
residue at position 35 of the peptide (Cys or SeCys) is critical
for the enzymatic transformation.
SbtM Removes Two Hydrogens from Cys35. To probe
the nature of the modification introduced by SbtM,
isotopologs of the SbtMa peptide were synthesized in which
Gly34 or Cys35 was replaced with [2,2 D2]-Gly or [3,3 D2]-
Cys, respectively. SbtM was incubated with each of the
isotopologs, and the resulting products were analyzed as
described above for the unlabeled substrate. The monoisotopic
peak of the +4 charge state envelope for the [2,2 D2]-Gly34
SbtMa peptide is at m/z of 1274.40 amu, which is within 1.7
ppm of the expected value (Figure 4B). Incubation with SbtM
leads to a 4.03 amu shift in the mass of peptide, which is
identical to that observed with the unlabeled substrate. In
contrast, the monoisotopic peak for the +4 charge state of the
SbtMa peptide substituted with [3,3 D2]-Cys35 (at 1274.40
amu) shifts to a m/z of 1272.89 amu. This corresponds to an
overall mass loss of 6.04 amu, indicating the loss of two
protons during the modification as well as two deuterium
atoms at the β-position of Cys35 (Figure 4B). These data
clearly implicate Cys35 as the site of modification.
SbtM Catalyzes H-Atom Abstraction from Cys35.
Radical SAM enzymes generally initiate catalysis by H-atom
abstraction from their respective substrate. Once we had
determined the Gly-Cys region of the peptide is the site of
chemistry, we examined H-atom transfer between these
residues and dAdo• with the deuterated isotopologs of the
substrate. Intriguingly, when SbtM is incubated with a peptide
containing [3,3 D2]-Cys35, the dAdo obtained from the
reaction is enriched in singly deuterated dAdo (Figure S8). By
contrast, dAdo from [2,2 D2]-Gly34 peptide does not show
any enrichment beyond that expected from natural abundance
of the isotope. The enrichment of singly deuterated dAdo in
the reactions containing the labeled Cys is consistent with H-
atom transfer between the β-carbon of Cys35 and dAdo•.
Collectively, these data show that at least two of the hydrogens
that are lost upon conversion of the substrate to the product
are derived from the β-carbon of Cys35.
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Figure 4. SbtM modifies SbtMa at position 35. (A) CID
fragmentation patterns of the modified and unmodified SbtMa
peptides show identical y18 fragments. By contrast, y20 fragments
show a 4 amu difference. Note that the y18 fragment is in the +1
charge state, whereas the y20 fragment is in the +2 state. (B) [3,3 D2]-
Cys35 SbtMa peptide undergoes a mass change of 6 amu in the
presence of SbtM. By contrast, [2,2 D2]-Gly34 undergoes a 4 amu
change upon modification. The measured monoisotopic features with
both variants are within 5 ppm of the expected values. Note that in
both cases, the envelope still shows some residual substrate.
NMR Characterization of Structure. To elucidate the
structure of the modified peptide, SbtMa-containing
[13C2,15N]-Gly34 and/or [13C3,15N]-Cys35 was synthesized
by SPPS purified by HPLC and lyophilized. The resulting
peptides were subjected to modification by SbtM and followed
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by treatment with iodoacetamide. The HPLC-purified
modified and carbamidomethylated peptides were resuspended
in buffer containing 10% D2O/H2O and analyzed by NMR.
We examined the fate of the amide protons of Gly34 and
Cys35 through a 15N-HSQC experiment, which correlates
amide protons to the amide nitrogen. The full NMR spectra
are shown in Figure S9. In a control experiment, unmodified
SbtMa isotopolog containing [13C2,15N]-Gly34 and [13C3,15N]-
Cys35, HN-Gly at (8.26, 110.4 ppm) and HN-Cys (8.26, 118.9
ppm) correlations are clearly observed (Figure 5A). After
incubation with SbtM, we observe correlations corresponding
to HN-Gly and HN-Cys from unreacted SbtMa, as well as a
single new correlation at (8.46, 110.3 ppm) assigned to Gly.
Significantly, we do not observe any modified peak for the
amide of Cys35, indicating that a structural change has
occurred, leading to the loss of the NH proton (Figure 5B).
To obtain additional structural information, a HNCACB
experiment was carried out to correlate the amide proton to
the adjacent carbon centers. With unmodified peptide
containing [ 13C2,15N]-Gly34 and [13C3,15N]-Cys35, the
previously assigned amide proton of Cys and Gly at 8.26
ppm (Figure 5A) both correlate with the α-carbon of Gly (45.4
ppm), through the peptide carbonyl. In addition, the amide
proton of the Cys residue shows a correlation to the α- and β-
carbons of the Cys residue at 55.75 and 36.06 ppm,
respectively (Figure 5C). We note that HNCys-βCys has a
negative intensity (denoted in red) as is expected of β-carbons
in HNCACB experiments.
Upon modification by SbtM, the HNCACB spectrum
obtained with a peptide containing [13 C2 ,15 N]-Gly34,
[13C3,15N]-Cys35 exhibits a single new correlation between
the amide proton of Gly at 8.47 ppm and the α-carbon of Gly
at 39.2 ppm (Figure 5D). The amide proton resonance at 8.47
ppm is similar to that observed in the 15N-HSQC of the
modified product (Figure 5B). Significantly, all of the
correlations to the amide proton of Cys are absent in the
modified peptide, consistent with the loss of the Cys amide
proton, which was also observed in the 15N-HSQC spectrum.
We note that an identical set of two-dimensional NMR
measurements was also made with SbtM-modified peptide
labeled with only [13C2,15N]-Gly34, which had not undergone
reaction with iodoacetamide. The only differences between
these data and the spectra presented in Figures S10 and S11
were small changes in chemical shifts. These data are included
in the Supporting Information. The loss of NH (Figure 5B)
and β-protons both in the HNCACB (Figure 5D) and the
deuterium labeling data (Figure 4B) strongly support the
notion that the modification catalyzed by SbtM involves
cyclization of the side chain in some fashion that also imparts
significant aromatic character to the product, as suggested by
the UV−visible spectrum of the modified peptide (Figure 3D).
Therefore, we also compared the 13C NMR spectra of
unmodified and SbtM-modified 13C and 15N-labeled SbtMa
peptides (Figure 5F). In addition to resonances from 13C
labeled atoms in the peptide, we also observe two additional
features at 76 and 29 ppm for DTT. The spectrum of the
modified peptide (see Figure S12) exhibits some of the same
features as the SbtM-modified peptide, as would be expected
from an unmodified peptide that remains in the sample. We
also observe several sharp resonances <100 ppm, presumably
from small molecules that are copurified in the course of the
workup. However, we observe three new resonances at 118,
120, and 165 ppm, all of which are in the aromatic region. This
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Figure 5. NMR analysis of the SbtM-modified peptide. The 15N-HSQC spectra of unmodified (A) and modified (B) SbtMa shows the loss of the
Cys amide proton. The HNCACB of unmodified (C) and modified (D) SbtMa confirm the loss of the Cys amide proton. (E) Panel shows
correlations in the unmodified peptide. (F) Panel shows the 13C spectra of the unmodified and modified SbtMa. The labels for the resonances in
the unmodified peptide are shown in cyan and those in the modified ones are shown in red.

observation will be examined further below but are consistent
with the formation of a conjugated system involving the Cys
side chain and amide.
FTIR of the Modified Peptide. Further investigation
toward the nature of the modification was carried out using
FTIR spectroscopy. The spectra of the unmodified and
modified SbtMa peptide were measured under vacuum in
KBr pellets (Figure 6). The unmodified SbtMa exhibits a
characteristic vS−H stretch at 2555 cm−1, which is attributed to
a thiol.43−46 This peak is not able to be resolved in the
modified peptide, though an additional feature at ∼2180 cm−1
is observed. The modification also introduces several new,
sharp features at around 1550 cm−1.
Additionally, the modified peptide also exhibits significant
changes in the amide II region. The secondary structures of the
unmodified or modified peptides are not known. However,
changes in the amide I and amide II regions are known to
reflect changes in the structure of the peptide backbone.47
Changes in the amide II region are proposed to result from
changes to the coupling of the backbone C−N stretches along
the peptide after cyclization of the Cys residue.48 Overall these
changes are indicative of the modification disrupting the
peptide backbone, similar to the effect of proline residues in
short peptides and proteins.43,49−51 However, since the three-
3355
dimensional structure of the peptides is not known, further
structural interpretation of the changes in the amide I and
amide II stretches is not possible.
Nature of the Modified SbtMa. In considering the
structure of the modified SbtMa, one needs to take into
account all of the MS, UV−visible, NMR, and FTIR
observations. SbtM catalyzes the loss of 4 amu from the
SbtMa peptide. The modification requires the presence of the
Cys/SeCys residue at position 35. Two of the hydrogen atoms
lost in this reaction are from the β-position of Cys/SeCys at
position 35 and the third from the corresponding amide
nitrogen. The product appears to retain an iodoacetamide-
sensitive group even after the modification by SbtM. The
location from which the fourth hydrogen lost is not known. We
know, on the basis of the H-transfer data, that at least one (and
possibly both) β-hydrogens of Cys/SeCys are transferred to
dAdo• forming dAdo in two successive H-atom transfer
events. Finally, the Fe content suggests the presence of
multiple [4Fe−4S] clusters, one of which, by analogy to other
radical SAM enzymes, we posit is involved in the activation of
SAM to generate the dAdo•, while at least one more may be
involved in the chemical transformation.
By analogy to anaerobic sulfatase maturing enzyme
(AnSME),52−54 we propose that SbtM catalyzes H-atom
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Figure 6. Analysis of SbtMa by FTIR. The experimental FTIR spectra
show the unmodified (black) and modified (red) SbtMa peptides.
Asterisks (*) represent new features present in the modified peptide.
Scaled features highlighting the S−H stretching region are shown in
the inset.
transfer from the β-carbon of Cys followed by oxidation of the
resulting species by a second cluster to form a thioformylgly-
cine intermediate (Figure 7). This intermediate could have
several fates. It could undergo cyclization with the carbonyl
Figure 7. Possible products of SbtMa peptide. The peptide is proposed to initially undergo H-atom transfer to dAdo to form a radical intermediate
that is oxidized to thioformylglycine. This thioformylglycine can be trapped in one of three manifolds to form either a 3-, 4-, or 5-membered
intermediate. Evidence for each is discussed in the text.
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moiety of the Gly-Cys amide bond to form a 5-membered ring.
Alternatively, it can form a 4-membered ring in an analogous
manner, with the oxygen from the carbonyl of Cys/SeCys, or, a
3-membered ring by cyclizing with the amide of the Gly-Cys
peptide bond. Each of these could subsequently undergo a
second H-atom transfer from the β-carbon of Cys/SeCys,
which upon oxidation would yield a thione product. Both the
5- and 3-membered ring products can undergo tautomerization
to present a thiol moiety. However, the tautomerization to the
aromatic thiooxazole (5-membered) is more favorable than the
tautomerized aziridine thione (3-membered), which would be
antiaromatic.
All of the initial products proposed in Figure 7 are isobaric
and could not be distinguished on the basis of MS. However,
we can eliminate the 4-membered ring from consideration
because it would still maintain the Cys amide, which is
inconsistent with the absence of a N−H resonance in the
HSQC data (Figure 5B) and because optimization by DFT
leads to a ring-opened product. DFT calculations, using a
truncated dipeptide model (see the Experimental Procedures),
suggest that the 5-membered ring containing product is more
stable than one containing a 3-membered ring (see Table S3).
Indeed, upon aromatization, the thiooxazole structure is 26
kcal/mol more stable than the aziridine thione. While the
inability to identify a resonance corresponding to the α-proton
of Cys strongly points to the aromatization of oxazoline to
oxazole, we wondered if a similar process could also occur with
the aziridine structure. DFT calculations suggest that the
analogous tautomerization of this structure is highly unfavor-
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Figure 8. 13C NMR characterization. 13C HSQC of the unmodified (A) and SbtM-modified (B) SbtMa peptide. The HMBC spectrum of the
modified SbtMa peptide zoomed in on the aromatic region (C) and the unmodified (D) and modified (E) structures with the pink dots
corresponding to the 13C HSQC correlations and the green arrow corresponding to the HMBC correlation.
able relative to the aziridine thione (by 42.4 kcal/mol), likely
because of the fact that the resulting structure is antiaromatic.
Both aziridine thione and thiooxazole products would be
reactive toward iodoacetamide. While the antiaromatic
structure would not be favored, resonance delocalization
from amide could lead to activation of thione for
carbamidomethylation. However, a key difference between
the aziridine thione and the thiooxazole structure is that they
will have differential reactivity toward mild reducing agents.
One would predict that aziridine thione would undergo
reduction by a hydride transfer reagent, whereas thiooxazole
would not.55 Therefore, we reduced SbtM-modified Cys-
SbtMa peptide with either NaBH4 or NaBD4 (Figure S14).
The data clearly show that the envelope of the charge states of
the product is identical to those of reactions that were not
quenched with either NaBH4 or NaBD4. This observation is
most consistent with the assignment of the modified amino
acid as a thiooxazole.
NMR spectroscopic observations lend further support for
the thiooxazole assignment. Perhaps the most crucial
observation is the fact that we see three resonances in 13C
NMR (Figure 5F) that are similar to those that have been
reported in other oxazole structures.56,57 These could be
assigned to the three carbons of oxazole (see Figure 7). To
obtain additional insights into the 13C NMR data, particularly
the 165 ppm resonance, a set of 13C HSQC spectra were
obtained with unmodified and SbtM-modified peptides (Figure
8). The chemical shift of the α-proton of Gly shifts from 3.9 to
4.2 ppm in the modified peptide. In the 13C-HMBC spectrum
of the modified peptide, these protons are correlated with the
165 ppm 13C resonance, directly support the connectivity of
the Gly-Cys, and the assignment of the 165 ppm feature to
what was the carbonyl carbon of the unmodified peptide. Since
the modification reaction was not complete, we also observed a
correlation between the carbonyl carbon of the Gly and the α-
protons of the unmodified peptide (at 3.8, 172 ppm).
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The UV−visible features attributed to the product further
support the assignment. The λmax = 304 nm feature in the UV−
visible data and its red shift to 313 nm in the SeCys-containing
SbtM-modified peptide are further suggestive of a conjugated
system (see Figure 3D). While unsubstituted oxazoles have
UV−visible features that are generally in the UV region,
substitutions that extend conjugation of the system lead to
shifting of the spectral feature.58
In addition to significant changes in the amide I and II
bands, which we have already discussed above, two additional
differences between the modified and unmodified FTIR
spectra are noteworthy. The modified peptide shows a new
band at 2150 cm−1, which is low for an S−H stretch. This
feature was probed via DFT analytical frequency calculations
of optimized unmodified and modified peptide models. Of the
potential modified products evaluated, only the calculated
spectrum of the thiooxazole species displayed an intense
vibration at 1985 cm−1 (Figure S15), originating from the S−H
stretching (Table S4). This predicted feature is somewhat
lower than normal S−H stretches because the optimized
structure reveals strong intramolecular hydrogen bonding,
which shifts this band to lower energy. The only other
dipeptide model that displays a feature in this region is 1H-
azirine-2-thiol (see Table S3), which DFT calculations show to
be 68.4 kcal/mol higher in energy than thiooxazole. Literature
precedents suggest that oxazoles would have strong features in
the IR in the region of 1500−1600 cm−1.5959 The spectra of
the modified SbtMa have features in this region of the FTIR
spectrum. While not diagnostic, these features are more
consistent with a thiooxazole product.
In summary, we have methodically considered all reasonable
structures that would account for the biochemical, chemical,
mass spectrometric, NMR, UV−visible, FTIR, and computa-
tional observations. The aromatic thiooxazole is the structure
that is most consistent with all of these observations.
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Figure 9. Mechanistic proposal for SbtM. SbtM catalyzes the oxidative cyclization of the Cys/SeCys residue in SbtMa. In this mechanism, two
successive H-atom transfer events are proposed, each of which is followed by oxidation via the auxiliary cluster. Note that the auxiliary cluster is
presumably reoxidized after each event to maintain an oxidized state.
■ DISCUSSION
The transformations that are catalyzed by members of the
radical SAM superfamily often have no parallels in polar
reaction spaces. It is perhaps not surprising that these enzymes
are often found in the biosynthetic pathways of natural
products because of their ability to catalyze challenging
reactions and introduce rare functional groups that bestow
selective advantage(s) to the producing organisms. SbtM is a
radical SAM enzyme that was first identified bioinformatically
in gene clusters that were presumed to be involved in the
biogenesis of SeCys-containing RiPP natural products. The
biological role of the mature SbtMa remains to be established.
A working mechanism for SbtM is shown in Figure 9.
Biochemical studies show that SbtM could support the
formation of at least two [4Fe−4S] clusters, one of which is
likely coordinated by the conserved radical SAM cluster
binding motif.4 By analogy to other SPASM domain-
containing radical SAM enzymes,21,24−30 one of the remaining
auxiliary clusters is likely proximal to the substrate in the active
site and is involved in the chemistry, whereas the last cluster
presumably plays an electron relay role. We propose that in the
first step, H-atom transfer occurs from the β-carbon of Cys/
SeCys at position 35 to the dAdo• to generate a substrate
radical intermediate. This intermediate is then oxidized by the
auxiliary cluster to form a thioformylglycine. This thioformyl-
glycine intermediate likely engages carbonyl oxygen and
undergoes cyclization with concomitant deprotonation of
amide nitrogen. The resultant O,S-acetal is oxidized to a
thione moiety by a H-atom transfer event likely from the β-
carbon of the Cys residue at position 35, followed by second
oxidation. This thione intermediate undergoes tautomerization
to form a stable aromatic thiooxazole. As discussed in the
results, multiple peptide products are possible; however, we
favor thiooxazole on the basis of the observations reported in
this manuscript. While oxazole moieties have been observed in
natural products, the mechanism by which SbtM catalyzes the
overall transformation is radically different. Oxazoles, such as
those in microcin B17, are formed in two steps that involve
activation of peptide carbonyl oxygen followed by crosslinking
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to the Ser residue. In the second step, a flavoenzyme
dehydrogenates the ring to form oxazole.60−63
The parallels between SbtM and cyclodehydratase/dehy-
drogenase systems are striking. Both function by activating the
substrate to present an electrophilic carbon center. However,
in SbtM’s case, it activates the β-carbon of a Cys/SeCys
residue by oxidation, whereas the cyclodehydratase activates
amide carbonyl carbon. In SbtM, the oxidation to oxazole is
predicted to also be carried out by SbtM using a second
equivalent of SAM. Therefore, SbtM dispenses with the two-
step pathway for oxazole formation by carrying out cyclization
and oxidation in the same active site using H-atom activation.
The auxiliary cluster of SbtM serves as a way station for the
reducing equivalents that are extracted from the substrate and
are presumably shuttled away from the enzyme through the
second auxiliary cluster.
In the nearly 20 years since the radical SAM superfamily was
described, the repertoire of reactions catalyzed by these
enzymes has expanded to include nearly every type of chemical
reactivity. Members of this ancient superfamily have been
discovered in biological niches that range from metabolism to
biosynthesis of natural products that confer a selective
advantage to the host. The physiological role, and in fact,
the identity of the natural product being produced by SbtM is
not known. However, the reaction uncovered here is significant
in several respects. The observation provides a parallel pathway
for oxazole formation in biology that relies on an ancient and
abundant iron−sulfur cofactor. The reaction catalyzed by
SbtM is potentially the first characterized modified SeCys-
containing RiPP natural product. The 53 amino acid peptide
characterized here should be considered a minimal substrate
for SbtM. Future studies with longer substrates, particularly
those that contain multiple potential modification sites, would
be important in understanding the scope and role of the
modification in nature. Finally, the mechanistic paradigm
uncovered in this manuscript extends the repertoire of
reactivity in the radical SAM superfamily.
https://doi.org/10.1021/acs.biochem.1c00469
Biochemistry 2021, 60, 3347−3361
Biochemistry
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pubs.acs.org/biochemistry
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biochem.1c00469.
Sequence alignment of the radical SAM enzyme (Figure
S1); codon-optimized gene sequence of SbtM (Figure
S2); protein sequence of the codon-optimized enzyme,
SbtM (Figure S3); SDS-PAGE analysis of SbtM (Figure
S4); mass spectrum of SbtMa peptide (Figure S5);
simulation of +4 charge state envelope (Figure S6); mass
spectrum of Cys35Ala and Cys35Ser SbtMa variants
(Figure S7); deuterium incorporation in 5′-deoxyade-
nosine (Figure S8); full NMR spectra of [13C2,15N]-
Gly34 [13C3,15N]-Cys35 SbtMa (Figure S9); 15N-HSQC
of [13C2,15N]-Gly34 SbtMa (Figure S10); full NMR
spectra of 15N-HSQC of [13C2,15N]-Gly34 SbtMa
(Figure S11); full 13C NMR spectra of unmodified
SbtMa (A) and modified (B) (Figure S12); full HMBC
spectrum of the SbtMa peptide (Figure S13); reduction
of SbtMa by sodium borohydride or sodium borodeu-
teride (Figure S14); DFT-calculated spectra of the
truncated dipeptide (Figure S15); ion observed in CID
fragmentation of modified (Table S1) and unmodified
SbtMa (Table S2); free-energy comparisons as calcu-
lated by DFT (Table S3); computational summary for
modified Gly-aziridine-2-thione dipeptide (Table S4);
DFT-optimized Cartesian coordinates of modified Gly-
aziridine-2-thione dipeptide (Table S5); computational
summary for modified Gly-1H-azirine-2-thiol dipeptide
(Table S6); DFT-optimized Cartesian coordinates of
modified Gly-1H-azirine-2-thiol dipeptide (Table S7);
computational summary for modified Gly-oxalone-5-
thiol dipeptide (Table S8); DFT-optimized Cartesian
coordinates of modified Gly-oxalone-5-thiol dipeptide
(Table S9); computational summary for modified Gly-
oxalone-5(4H)-thione dipeptide (Table S10); DFT-
optimized Cartesian coordinates of modified Gly-
oxalone-5(4H)-thione dipeptide (Table S11); computa-
tional summary for modified Gly-oxalone-5(2H)-thione
dipeptide (Table S12); DFT-optimized Cartesian
coordinates of modified Gly-oxalone-5(2H)-thione
dipeptide (Table S13); computational summary for
modified Gly-azirine-2-thiolate dipeptide (Table S14);
and DFT-optimized Cartesian coordinates of modified
Gly-azirine-2-thiolate dipeptide (Table S15) (PDF)
Accession Codes
SbtM, WP_083768621.
■ AUTHOR INFORMATION
Corresponding Author
Vahe Bandarian − Department of Chemistry, University of
Utah, Salt Lake City, Utah 84112, United States;
orcid.org/0000-0003-2302-0277; Email: vahe@
chem.utah.edu
Authors
Julia K. Lewis − Department of Chemistry, University of Utah,
Salt Lake City, Utah 84112, United States
Andrew S. Jochimsen − Department of Chemistry, University
of Utah, Salt Lake City, Utah 84112, United States
Sarah J. Lefave − Department of Chemistry, University of
Utah, Salt Lake City, Utah 84112, United States
3359
Article
Anthony P. Young − Department of Chemistry, University of
Utah, Salt Lake City, Utah 84112, United States;
orcid.org/0000-0003-2273-346X
William M. Kincannon − Department of Chemistry,
University of Utah, Salt Lake City, Utah 84112, United
States
Andrew G. Roberts − Department of Chemistry, University of
Utah, Salt Lake City, Utah 84112, United States
Matthew T. Kieber-Emmons − Department of Chemistry,
University of Utah, Salt Lake City, Utah 84112, United
States; orcid.org/0000-0002-6357-5579
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biochem.1c00469
Author Contributions
The manuscript was written through the contributions of all
authors.
Funding
V.B. is supported by the National Institute of General Medical
Sciences of the National Institutes of Health by the grant R35
GM126956. M.T.K.-E. is supported by Physical Biosciences
Core Research Activity in the Chemical Sciences, Geosciences,
and Biosciences Division of the Basic Energy Sciences Program
of the Department of Energy (DE-SC0020954).
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The NMR measurements were recorded at the David M. Grant
NMR Center, which is a University of Utah Core Facility. The
funding for the construction of the Center and the helium
recovery system were obtained from the University of Utah
and the National Institutes of Health awards 1C06RR017539-
01A1 and 3R01GM063540-17W1. The NMR instruments
were purchased with the support of the University of Utah and
the National Institutes of Health awards 1S10OD25241-01.
We are indebted to Dr. Peter Flynn for assistance with the
NMR measurements.
■
ABBREVIATIONS
BSA, bovine serum albumin; CID, collision-induced dissocia-
tion; CV, column volume; dAdo•, 5′-deoxyadenosine radical;
dAdo, 5′deoxyadenosine; DFT, density functional theory; dT,
dithionite; DTT, dithiothreitol; DNTP, dithiobis-5-nitro-
pyridine; Fmoc, flurenylmethyloxycarbonyl; FTIR, Fourier-
transform infrared spectroscopy; Gnd·HCl, guanidinium
hydrochloride; HPLC, high-performance liquid chromatog-
raphy; HSQC, heteronuclear single quantum coherence; ICP-
MS, inductively coupled plasma-mass spectrometry; IPTG,
isopropyl β-D-1-thiogalactopyanoside; KPi, potassium phos-
phate; LB, Lennox broth; Mob, methoxybenzyl; NMR, nuclear
magnetic resonance; 5-Npys, 2-thio(5-nitropyridyl); orf, open
reading frame; PIPES, 1,4-piperazinediethanesulfonic acid;
PMSF, phenylmethanesulfonyl fluoride; Q-Sepharose, quater-
nary ammonium Sepharose; RiPP, ribosomally synthesized
post-translationally modified peptide; SAM, S-adenosyl-L-
methionine; SDS-PAGE, sodium dodecyl sulfate polyacryla-
mide gel electrophoresis; SECIS, selenocysteine insertion
sequence; SPPS, solid-phase peptide synthesis; TCA, trichloro-
acetic acid; TCEP, tris(2-carboxyethyl) phosphine; TEV,
tobacco etch virus; Tris, tris(hydroxymethyl)aminomethane
https://doi.org/10.1021/acs.biochem.1c00469
Biochemistry 2021, 60, 3347−3361
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