Thioether crosslinkages created by a radical SAM enzyme.
ABSTRACT Unusually versatile: While the β-carbon thioether linkage in lantibiotics has long been appreciated and is relatively well characterized, a recent publication shows that the unusual sulfur-to-α-carbon thioether crosslinks in subtilosin A are produced by a radical SAM enzyme, AlbA, that contains two [4 Fe-4 S] clusters, thus highlighting the versatility of post-translational modifications in natural product biosynthesis.
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ABSTRACT: Enzymes in the radical SAM (RS) superfamily catalyze a wide variety of reactions through unique radical chemistry. The characteristic markers of the superfamily include a [4Fe-4S] cluster coordinated to the protein via a cysteine triad motif, typically CX(3)CX(2)C, with the fourth iron coordinated by S-adenosylmethionine (SAM). The SAM serves as a precursor for a 5'-deoxyadenosyl radical, the central intermediate in nearly all RS enzymes studied to date. The SAM-bound [4Fe-4S] cluster is located within a partial or full triosephosphate isomerase (TIM) barrel where the radical chemistry occurs protected from the surroundings. In addition to the TIM barrel and a RS [4Fe-4S] cluster, many members of the superfamily contain additional domains and/or additional Fe-S clusters. Recently characterized superfamily members are providing new examples of the remarkable range of reactions that can be catalyzed, as well as new structural and mechanistic insights into these fascinating reactions.Current Opinion in Structural Biology 11/2012; · 8.74 Impact Factor
Thioether Crosslinkages Created by a Radical SAM Enzyme
Qi Zhang*[a, b]and Yi Yu*[a]
Recent years have seen an upsurge in the discovery
of natural products that are ribosomally synthesized
and post-translationally modified. The vast majority
of these peptide compounds are potent antimicrobi-
als that might provide the hosts with competitive
advantages in complex microbial niches. According-
ly, they represent a large class of antibiotics with
promise for clinic and industrial use. Although ribo-
somes cannot explore building blocks beyond the
canonical 20 proteinogenic amino acids, the nascent
peptides can be extensively tailored, and this leads
to a highly diverse array of architectures that are
comparable to those produced by nonribosomal
The thioether crosslinks in lantibiotic biosynthesis
are among the most prominent post-translational
modifications.The formation of these sulfur-to-b-
carbon thioether linkages in lantibiotics is relatively
well understood and involves initial dehydration of
Ser and Thr residues and subsequent intramolecular
Michael-type addition of Cys thiols to the newly
formed dehydroamino acids (Scheme 1A).In contrast to the
prevalent b-carbon thioether linkages, sulfur-to-a-carbon thio-
ether bonds are relatively rare in nature. One example of such
a thioether bond is found in cyclothiazomycin,a member of
the thiopeptide antibiotic family, which is of ribosomal origin.
The a-carbon thioether bond of cyclothiazomycin links the C-
terminal Cys to a dehydroalanine residue and forms a second
macrocyclic ring; this makes the scaffold distinct from those of
other thiopeptide family members.Although the mechanism
for this thioether linkage is unclear, an acyl imine intermediate
might be involved in the process. Nucleophilic attack of the
Cys thiol on the imine intermediate could install the unusual
thioether bond (Scheme 1B). Similar imine chemistry has been
proposed recently for the dealkylation reaction in nosiheptide
However, another group of ribosomal natural products that
contain sulfur-to-a-carbon thioether bonds has emerged in
recent years, it includes the head-to-tail-cyclized peptide subti-
losin A,the Bacillus sporulation killing factor (SKF),thuricin
H,and two closely related peptides of thuricin CD.These
peptide compounds have been classified as sactibiotics (sulfur-
to-a-carbon antibiotic).Unlike cyclothiazomycin, sactibiotics
usually contain multiple thioether bonds, and the hydroxyl
groups of Ser/Thr side chains remain intact after thioether link-
age (Scheme 1C), thus indicating that a different biosynthetic
mechanism could be involved. Characterization of the subtilo-
sin A biosynthetic gene cluster revealed a radical S-adenosyl-
methionine (SAM) enzyme, AlbA, that was shown by mutation-
al analysis to be essential for subtilosin A production.In ad-
dition, AlbA homologues are present in the biosynthetic gene
clusters of all other sactibiotics.Radical SAM enzymes usually
share a characteristic CXXXCXXC motif—for binding a [4Fe?
4S] cluster—that reductively cleaves SAM to generate a highly
reactive 5’-deoxyadenosyl (dAdo) radical. Radical chemistry is
then imposed on a variety of organic substrates, and this leads
to a highly diverse array of reactions relevant to nucleic acid
and protein modification, and the biosynthesis of vitamins,
coenzymes and antibiotics.Given that radical SAM enzymes
are often involved in catalyzing chemically unusual and com-
plex biotransformations,[12d]AlbA could be a suitable candidate
for forming the unusual a-carbon thioether bonds in subtilo-
sin A. This activity of AlbA (Scheme 1C) has now been demon-
strated in vitro by Marahiel and colleagues,and this high-
lights the versatility of post-translational modifications in natu-
ral product biosynthesis and further expands the catalytic rep-
ertoire of radical SAM superfamily enzymes.
Scheme 1. Thioether linkages in natural product biosynthesis. A) b-carbon thioether
bond formation catalyzed by lantibiotic synthase. B) Proposed mechanism for the instal-
lation of the thioether bond in cyclothiazomycin biosynthesis. C) Sulfur-to-a-carbon thio-
ether linkage catalyzed by AlbA during subtilosin A maturation. X1, X2and X3represent
different linker regions.
[a] Prof. Dr. Q. Zhang, Prof. Dr. Y. Yu
Key Laboratory of Combinatory Biosynthesis and
Drug Discovery (Ministry of Education) and
School of Pharmaceutical Sciences, Wuhan University
185 East Lake Road, Wuhan 430071 (P. R. China)
[b] Prof. Dr. Q. Zhang
State Key Laboratory of Bioorganic and Natural Products Chemistry
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences
345 Lingling Road, Shanghai 200032 (P. R. China)
ChemBioChem 2012, 13, 1097–1099? 2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim
In a beautifully executed series of biochemical and
MS analyses, the authors showed that AlbA is indeed
the enzyme that builds the three thioether bridges
on the precursor peptide SboA. That this reaction
occurs only when the leader peptide is present is
consistent with the leader peptide’s indispensable
role in directing post-translational tailoring reactions,
as found in the biosynthetic pathways of other ribo-
somal natural products.The results also indicated
that the thioether linkage is the first step in subtilosi-
n A maturation. Extensive spectroscopic and muta-
tional studies showed that, unexpectedly, AlbA has
an additional C-terminal [4Fe?4S] cluster to the one
binding to the canonical N-terminal CXXXCXXC motif.
Both of the [4Fe?4S] clusters are essential for thio-
ether bond formation, whereas enzymes with only an
N-terminal [4Fe?4S] cluster are still able to cleave
SAM, albeit with a reduced activity.
Several radical SAM enzymes contain more than
one Fe?S cluster. Biotin synthase BioB,lipoyl syn-
thase LipA,and the methylthiotransferases MiaB
and RimOcontain additional [2Fe?2S] clusters that
can serve as the sulfur source for radical-mediated
sulfur-insertion reactions. For the dehydrogenase
BtrNand anaerobic sulfatase maturase AtsB,ad-
ditional [4Fe?4S] clusters are provided for binding
the substrates and serving as one-electron oxidants.
Although it was shown that the C-terminal [4Fe?4S]
cluster of MoaA, which is involved in molybdopterin
biosynthesis, binds the substrate guanosine triphos-
phate in the crystal structure,the exact role of this
cluster in catalysis is largely elusive. The role of the C-
terminal [4Fe?4S] cluster of AlbA seems similar to that of BtrN
and AtsB, as detailed UV–visible spectroscopic analysis clearly
showed the interaction between the C-terminal [4Fe?4S] clus-
ter and the precursor peptide SboA in a leader-peptide-depen-
dent manner. A working hypothesis for the mechanism of
AlbA catalysis has been proposed (Scheme 2A),which
seems to be a combination of the catalysis of isopenicillin N
synthase(Scheme 2B) and BtrN/AstB (Scheme 2C). Owing to
the propensity of the Fe?S bond to cleavage homolytically,
binding of the thiol group to the ferric ion or the oxidized
[4Fe?4S] cluster may be viewed as production of an inert thiyl
radical, which could couple with the carbon-centered radical
to form the thioether bond (Scheme 2A and B). In
regard to this scenario, it would be easy to conclude
that AlbA cannot form the ether linkage, as homolyt-
ic cleavage of the Fe?O bond will not occur in the re-
action owing to the high electronegativity of the
oxygen atom. Consistent with this, by using an in
vivo production system, Marahiel and co-workers
showed that, except for the Phe-to-Tyr mutant, no
SboA mutants (including the three Cys-to-Ser mu-
tants) could be processed to the mature products.
Chemical derivatization coupled with MS analysis
in time-dependent assays also provided interesting
insights into enzyme catalysis. The assays showed
that the fully thioether-bridged species was produced in
a time-dependent manner, whereas relatively little of the par-
tially bridged species was observed. This suggested processive
action of the enzyme. Similar results were observed for the de-
hydratase activity of lantibiotic synthase,thus suggesting
that the catalytic processivity might be common for the matu-
ration of ribosomal natural products, for which multiple
rounds of catalysis on the precursor peptide are usually re-
quired. Intriguingly, the five known sactibiotics all possess
a ladder-like topology (Scheme 3; SKF contains only one thio-
ether bond and an additional disulfide bond, but these two
crosslinks still form a ladder-like structure). This sactibiotic
topology is in distinct contrast to that of lantibiotics, which
Scheme 2. A) Proposed mechanism for AlbA catalysis. The N- and C-terminal [4Fe?4S]
clusters are shown as orange and green spheres, respectively. B) Thioether linkage by iso-
penicillin N synthase. C) A representative scheme for dehydrogenation reactions cata-
lyzed by BtrN or AstB.
Scheme 3. The proposed zipper mechanism for AlbA catalysis. The orange and green
spheres represent the two [4Fe?4S] clusters of the enzyme.
? 2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2012, 13, 1097–1099
Q. Zhang and Y. Yu
usually have much more complex and irregular rings. Through
detailed kinetic analyses of different SboA mutants, the au-
thors showed that formation of the first thioether bond assists
the formation of the second one involving a proximate cys-
teine residue. Thus during catalysis, AlbA might “zip” together
(Scheme 3). This mechanism fits the ladder-like topology of the
final product and could be common for the biosynthesis of
The results presented by Marahiel and colleagues show the
intriguing chemistry of AlbA and the versatility of post-transla-
tional modifications in general. Recent metagenomic analysis
has revealed the presence of AlbA-like radical SAM enzymes in
a variety of metagenomic environments, thereby suggesting
a widespread distribution of sactibiotic-encoding gene clusters
in nature.The characterization of AlbA might thus lay the
groundwork for future efforts to identify new members of this
burgeoning class of natural products. Together with the re-
cently established biosynthetic paradigms of several ribosomal
natural products, including thiopeptides, cyanobactins, micro-
viridins, and S-linked glycopeptides,[14, 24]it seems that the di-
versity of the ribosomal world is just beginning to be appreci-
ated. Given the vast tracts of fertile ground discovered as a
result of the ever-increasing genomic information, future stud-
ies on ribosomal natural products are promising, particularly at
a time when there is an urgent need for new antibiotics.
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Received: March 21, 2012
Published online on May 3, 2012
ChemBioChem 2012, 13, 1097–1099? 2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim
Catalytic Mechanism of AlbA