Identification of a Pyrrole Intermediate Which Undergoes C-Glycosidation and Autoxidation to Yield the Final Product in Showdomycin Biosynthesis

Article abstract of DOI:10.1002/anie.202105667

Showdomycin is a C-nucleoside bearing an electrophilic maleimide base. Herein, the biosynthetic pathway of showdomycin is presented. The initial stages of the pathway involve non-ribosomal peptide synthetase (NRPS) mediated assembly of a 2-amino-1H-pyrrole-5-carboxylic acid intermediate. This intermediate is prone to air oxidation whereupon it undergoes oxidative decarboxylation to yield an imine of maleimide, which in turn yields the maleimide upon acidification. It is also shown that this pyrrole intermediate serves as the substrate for the C-glycosidase SdmA in the pathway. After coupling with ribose 5-phosphate, the resulting C-nucleoside undergoes a similar sequence of oxidation, decarboxylation and deamination to afford showdomcyin after exposure to air. These results suggest that showdomycin could be an artifact due to aerobic isolation; however, the autoxidation may also serve to convert an otherwise inert product of the biosynthetic pathway to an electrophilic C-nucleotide thereby endowing showdomycin with its observed bioactivities.

Full text of DOI:10.1002/anie.202105667

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Accepted Article
Title: Identification of a Pyrrole Intermediate Which Undergoes C-
Glycosidation and Autoxidation to Yield the Final Product in
Showdomycin Biosynthesis
Authors: Daan Ren, Minje Kim, Shao-An Wang, and Hung-wen Liu
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RESEARCH ARTICLE
Identification of a Pyrrole Intermediate Which Undergoes C-
Glycosidation and Autoxidation to Yield the Final Product in
Showdomycin Biosynthesis
Daan Ren, Minje Kim, Shao-An Wang and Hung-wen Liu*
[*]
D. Ren, M. Kim, Dr. S.-A. Wang, Prof. Dr. H.-w. Liu
Department of Chemistry, and Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, University of Texas at Austin
Austin, TX 78712 (USA)
E-mail: h.w.liu@mail.utexas.edu
Supporting information for this article is given via a link at the end of the document.
Abstract: Showdomycin is a C-nucleoside bearing an electrophilic
maleimide base. Herein, the biosynthetic pathway of showdomycin is
presented. The initial stages of the pathway involve non-ribosomal
peptide synthetase (NRPS) mediated assembly of a 2-amino-1H-
pyrrole-5-carboxylic acid intermediate. This intermediate is prone to
air oxidation whereupon it undergoes oxidative decarboxylation to
yield an imine of maleimide, which in turn yields the maleimide upon
acidification. It is also shown that this pyrrole intermediate serves as
the substrate for the C-glycosidase SdmA in the pathway. After
coupling with ribose 5-phosphate, the resulting C-nucleoside
undergoes a similar sequence of oxidation, decarboxylation and
deamination to afford showdomcyin after exposure to air. These
results suggest that showdomycin could be an artifact due to aerobic
isolation; however, the autoxidation may also serve to convert an
otherwise inert product of the biosynthetic pathway to an electrophilic
C-nucleotide thereby endowing showdomycin with its observed
bioactivities.
Figure 1. C-Ribosylnucleoside antibiotics.
How the C-nucleosides are assembled in nature has been a
subject of current interest. Recently, the C-glycosylation reactions
in the formycin A (2) and pyrazofurin (3) biosynthetic pathways
were shown to be catalyzed by ForT and PyfQ, respectively.[
Both utilize phosphoribosyl pyrophosphate (PRPP) as the ribose
donor, and the reactions proceed via electrophilic aromatic
substitution.[14,16] In contrast to ForT and PyfQ, however, early
studies of pseudouridine (4) biosynthesis showed that
pseudouridine monophosphate synthase (YeiN) catalyzes C-
glycosidic bond formation and cleavage between ribose 5-
14-16]
Introduction
Showdomycin (1) along with formycin A (2), pyrazofurin (3),
pseudouridine (4), alnumycin C1 (5) and minimycin (6) are C-
nucleosides with structures characterized by a C‒C ribosyl
linkage between ribofuranose and a heterocyclic base (Figure 1).
[
1-4]
Showdomycin (1) isolated from Streptomyces showdoensis [5,
6
]
phosphate (14, R5P) and uracil through a ribose ring-opening
is known to inhibit DNA and RNA polymerases isolated from
mechanism.[
17,18]
Likewise, AlnA in alnumycin C1 (5) biosynthesis
Escherichia coli leading to antibacterial and anti-tumor activities.
[
7-10]
exhibits moderate homology to YeiN and appears to catalyze the
attachment of R5P to the polyketide aglycone via a mechanism
involving an enediolate intermediate.[19,20] Finally, the gene cluster
for showdomycin biosynthesis (sdm) was recently identified in S.
showdoensis by screening for homologs of the C-glycosidase
AlnA.[21] The corresponding C-glycosidase in showdomycin
pathway has thus been hypothesized to be the gene product
SdmA, and the observation that deletion of the sdmA gene
prevents showdomycin biosynthesis in vivo is consistent with this
hypothesis.[21] Nevertheless, this gene assignment has not been
tested in vitro such that the biological assembly of showdomycin
remains to be definitively established.
It was also found to inactivate the nucleoside uptake system,
and this inhibition can be reversed by nucleoside and thiol
compounds such as cysteine.[11,12] It was therefore suggested that
the bioactivity of showdomycin is a consequence of its maleimide
moiety, which likely acts as a Michael acceptor and thus an
alkylating agent when it undergoes nucleophilic addition by a
sulfhydryl group on its inhibitory targets.[11,12] On the basis of its
reactivity towards thiols, showdomycin and its synthetic
derivatives have also been designed as probes for the labeling
and identification of clinically important enzymes in pathogenic
bacteria.[13]
Results and Discussion
1
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RESEARCH ARTICLE
To address these unresolved questions, the sdm gene cluster
was analyzed, and a cassette consisting of sdmC, D, E, F and G
was found to be homologous to five genes in the kosinostatin (8)
biosynthetic gene cluster (kstB1, B2, B3, B4 and B6, respectively)
SdmE, a homolog of L-ectoine synthase (EctC). Canonical EctC
is responsible for the conversion of 17 to L-ectoine (18, see Figure
3A)[ and can also catalyze the condensation of 9 to 10 as a side
reaction.[24] Ant24 is a homolog of SdmE that is similarly believed
to catalyze the same reaction (i.e., 9 → 10, see Figure 3B) during
the biosynthesis of anthelvencin A (19) according to gene deletion
and chemical complementation assays by feeding the mutant with
10.[25] Indeed, when 10 μM purified SdmE (Figure S1) was
incubated with 5 mM L-glutamine in tris(hydroxymethyl)amino-
23]
(
Figures 2A and 2B).[22] Showdomycin (1) and kosinostatin (8) are
both characterized by a moiety having a pyrrolidine skeleton (see
ring F in 8), and it was therefore hypothesized that construction of
this moiety (e.g., 7/12 in Figure 2C) is catalyzed by the gene
products of the two homologous cassettes shared by each gene
cluster. This hypothesis also implies that the early steps of
showdomycin biosynthesis involve nonribosomal peptide
synthesis, because sdmC and sdmD (kstB1 and kstB2) are
annotated as encoding nonribosomal peptide synthetases
methane (Tris) buffer (pH 8.0) and the reaction followed by 1
H
NMR, new signals were observed consistent with a synthetically
prepared standard of 10 (see Figure 3C) in support of this
hypothesis. This observation is also consistent with the report that
none of the 20 proteinogenic amino acids can be uploaded onto
SdmD by SdmC.[ Thus, the conversion of L-glutamine (9) to 10
appears to be necessary to provide the proper substrate form
recognized by the SdmD/SdmC NRPS system.
(
NRPS). SdmG, which is annotated as a transglutaminase-like
21]
protein, may then catalyze hydrolysis of the thioester linkage to
release the free acid 13 as the substrate for the C-glycosidase
SdmA. Subsequent tailoring steps including dephosphorylation,
deamination, oxidation and decarboxylation would then lead to
maturation of the nucleobase in showdomycin (1).
A
B
A
sdm gene cluster:
P
O
B
A
N M L K J
Thioesterase
C
D E
F
G
H I
Flavin-dependent
oxidase
C-glycosidase
Phosphatase
NRPS
Deaminase
L-ectoine synthase
C
Transporter
Regulator
Others
(
(
(
i) 10 standard
H-3
B
kst gene cluster:
H-4
H-5
H-4ꢀ
…
…
…
…
ii) Full reaction
iii) No enzyme
B1 B2 B3 B4
B6
C
δ (ppm)
D
E
1
2
SdmC+F
SdmC
11
holo-SdmD
No enzyme
Retention time (min)
Figure 3. (A) EctC-catalyzed condensation of 17 to L-ectoine (18). (B)
Biosynthesis of anthelvencin A (19) involving cyclization of L-Gln (9) to 10 by
Ant24. (C) Monitoring product formation in the SdmE catalyzed reaction using
1
Figure 2. (A) Biosynthetic gene cluster of showdomycin in S. showdoensis. (B)
Partial gene cluster of kosinostatin (8). The F ring was proposed to be
biosynthesized from precursor 7.[22] (C) Proposed biosynthetic pathway of
showdomycin (1).
H NMR spectroscopy. (D) HPLC analysis of the SdmC and SdmF catalyzed
reactions. holo-SdmD coelutes with 12. (E) Ejection of Ppant-containing ions by
MS fragmentation.
[26]
To test this hypothesis, 50 μM apo-SdmD was incubated with
A
possible biosynthetic pathway for showdomycin
1
mM 10 in the presence of
phosphopantetheinyl transferase (Sfp), 1 mM ATP, 75 μM
coenzyme A, 10 mM MgCl and mM tris-(2-
2
μM SdmC,
2
μM
biosynthesis can thus be envisaged in which a protein-tethered 2-
aminopyrrole species 12 (7 in the kosinostatin pathway) is
generated via the actions of SdmE/C/D/F and serves as a key
intermediate (see Figure 2C). The proposed pathway is likely
initiated with the cyclization of L-glutamine (9) to 10 catalyzed by
2
5
carboxyethyl)phosphine (TCEP) in 100 mM Tris buffer (pH 8.0)
for 2 h. After quenching the reaction with acetonitrile, HPLC
analysis of the supernatant revealed the presence of a new
2
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RESEARCH ARTICLE
peptidyl carrier protein species (Figure 3D). To track any change
at the acylation carrier site of its phosphopantetheinyl (Ppant) arm,
ESI-MS was employed to detect the Ppant ion fragment ejected
from the intact protein (see Figure 3E and supporting information).
Indeed, an MS signal was observed for an ion fragment having
the mass expected for Ppant modified with 11 (Figure S2). This
implies that showdomycin biosynthesis begins with formation of
the modified SdmD peptide-carrier-protein following cyclization of
L-glutamine (9 → 10 → 11).
A
B
3
52 nm
Control
3
0 s
0 s
0 s
Wavelength (nm)
0 min
6
2
0 min
0 min
0 min
120 min
9
4
6
24 h
3
10 nm
Formation of a 2-aminopyrrole from the 2-aminopyrroline
intermediate 11 requires an oxidation reaction that may be
catalyzed by SdmF, which is annotated as a flavin-dependent
oxidase. When 1 μM SdmF was included in the above assay
under aerobic condition, a new species was observed by MS with
a molecular weight two mass units less than 11 (Figures 3D and
S2). Purification of the SdmD/SdmC/SdmF coupled reaction
mixture by gel filtration led to isolation of the modified SdmD,
which exhibited a UV-Vis absorption maximum at 352 nm at pH
Wavelength (nm)
Wavelength (nm)
C
8
.0 (Figure S3). A similar spectrum with a maximum absorbance
at 350 nm (pH 8.0) was also observed with a synthesized N-
acetylcysteamine (S-NAc) analog of 12 (Figure S3), suggesting
that the dehydrogenated product formed in the presence of SdmF
is the proposed intermediate 12, which contains a 2-aminopyrrole
moiety conjugated to a thioester. Furthermore, when SdmF was
incubated with 10 in the absence of SdmC and SdmD, no
D
1
substrate consumption was observed by H-NMR spectroscopy
P2
(
Figure S4). These observations imply that SdmF catalyzes the
Coeluted
1
2
dehydrogenation of 11 to 12 in a reaction that takes place only
after loading of 10 onto the peptide-carrier-protein SdmD.
Incubation of the purified 12 with SdmG resulted in the
decrease of the absorbance at 352 nm and an increase in
absorbance at 288 nm with an isosbestic point at 310 nm (Figure
P1
5
Coinjection of traces 3 & 4
holo-SdmD + maleimide
+SdmG_3 h
4
3
2
1
4
A). Therefore, it was hypothesized that SdmG catalyzes the
+
SdmG_5 min
2
3
subsequent hydrolysis of 12 to generate the free 2-amino-1H-
pyrrole-5-carboxylic acid 13. However, LC-MS analysis failed to
detect 13 following deproteinization of the reaction mixture via
ultrafiltration, and the absorbance at 288 nm was also found to
diminish over time (Figure 4B). One possible explanation for the
results with SdmG and intermediate 12 is that 13 is unstable
under aerobic conditions, because 2-aminopyrroles are prone to
No enzyme
2
4
Retention time (min)
Figure 4. (A) Time-course of the SdmG-catalyzed reaction monitored by UV-
Vis spectroscopy. The final enzyme and substrate concentrations were 50 nM
and 30 μM, respectively. The inset shows difference spectra calculated by
subtracting the spectrum of a control with no SdmG from each time-point. (B)
Decomposition of the SdmG product determined by the diminishing absorbance
at 288 nm of the reaction filtrate over 24 h. (C) The proposed autoxidation of 13.
Decomposition of the endoperoxide intermediate 20 to 21 and 22 may proceed
via either direct decarboxylation (route a) or O-O bond cleavage triggered by
imine formation (route b). (D) HPLC analysis of the proteinaceous products from
the aerobic SdmG reaction. The elution was monitored at 352 nm (dash line) to
determine the consumption of substrate 12.
autoxidation.[27-29]
Moreover,
the
pyrrole-2-carboxylate
[30]
intermediates identified in the biosynthesis of violacein
and
rebeccamycin[
31,32]
have also been shown to react with oxygen
resulting in oxidative decarboxylation to the corresponding oxo-
products (Figure S5). These early reports suggested that 13 may
undergo a similar autoxidation and subsequent decarboxylation
under aerobic conditions to yield 2-iminopyrrolone 21 and
maleimide 22 following SdmG catalyzed hydrolysis of 12 (Figures
4
B and 4C). Given that 21 and 22 are potential Michael acceptors
and holo-SdmD would also be released in the SdmG reaction, the
free thiol of the phosphopantetheinyl arm of holo-SdmD may in
turn react with 21 or 22 to form adducts such as 23 or 24 (Figure
In order to test these hypotheses, the SdmG assay was again
performed and analyzed by LC-MS to investigate the formation of
modified peptidyl carrier proteins. HPLC analysis revealed the
complete disappearance of substrate 12 within 5 min as indicated
by the absence of any signal at 352 nm (Figure 4D trace 2).
Meanwhile, gradual formation of two new peaks P1 and P2 over
4
D).
3
h were found as the major SdmD-bound species in the SdmG
reaction (Figure 4D). MS analysis of the Ppant ejection ions of P1
and P2 were consistent with the proposed structures of 23 and 24
(
Figure S6). Furthermore, a standard of 24 prepared by reacting
maleimide with holo-SdmD was observed to coelute with P2
produced in the SdmG catalyzed reaction (Figure 4D trace 5).
3
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RESEARCH ARTICLE
Finally,
when
15N
2
-L-glutamine
was
used
in
the
annotated as a C-glycosidase in the sdm cluster. To test this
hypothesis, the in vitro characterization of SdmA using ribose 5-
phosphate (R5P, 14) as the electrophile was conducted under
anaerobic conditions to prevent autoxidation of 13. First, 13 was
freshly prepared by incubating 12 with SdmG and then removing
the enzymes by ultrafiltration. When 10 μM SdmA was added to
the resulting solution containing 13 (ca. 80 μM) along with 0.8 mM
SdmE/SdmC/SdmD/SdmF/SdmG reaction, an increase of two
mass units of the Ppant ejection ion during LC-MS analysis of P1
was observed consistent with the assigned structure 23 (Figure
S7). Therefore, the observed consumption of substrate 12 and
formation of 23 and 24 agrees with the proposed reaction
sequence in which 13 is slowly oxidized and covalently trapped
by holo-SdmD.
2
R5P (14) and 5 mM MgCl , a new species was detected by HPLC
with a UV-vis absorption spectrum nearly identical to that of 13
but with a different HPLC retention time (Figures 6B and S9).
HRMS analysis of this new product revealed a chemical
A
B
16 2 9
composition of C10H N O P, which is consistent with the
structure of 15 (calc. 339.0593, found 339.0599). The in-source
decarboxylated fragment was also observed by MS (calc. m/z for
2
3: Exact mass: 357.1591
24: Exact mass: 358.1431
+
C
9
H
16
N
2
O
7
P [M-CO
2
] 295.0695, found 295.0710) (Figure S10),
18O
2
18
O2
indicating the presence of a labile carboxyl group and supporting
the assignment of the SdmA product as 15. However, similar
results would also be expected for a product containing a C4-C1'
linkage (i.e., 27) rather than a C3-C1' linkage as shown in 15
(Figure 6A). While the isolation and direct characterization of 15
failed due to its instability, the regiochemistry of adduct formation
+
2 Da
+
2 Da
H218O
H218O
2
3
24
2
was determined by using the [4- H]-isotopolog of 13 prepared
2
enzymatically from [3,3- H
2
]-L-glutamine. LC-MS analysis of the
m/z
m/z
corresponding SdmA product showed retention of one deuterium
see Figure S11), indicating 15 rather than 27 is the enzymatic
C
D
(
2
3
24
product of SdmA. This regioselectivity is also consistent with the
increased nucleophilicity of C3 of 13 due to the amino substituent
at the C2 position. Moreover, if SdmB, which is annotated as a
phosphatase, was present in the reaction, the correct mass of the
dephosphorylated product 16 was detected (calc. m/z for
w/o acid treatment
23
w/ acid treatment
m/z
m/z
+
C H N O [M+H] 259.0930, found 259.0923) (Figure S10).
1
0
15
2
6
Interestingly, unlike the cases of ForT and PyfQ, PRPP is not a
substrate for SdmA (Figure 6B). It was also found that maleimide
(22) could not be processed by SdmA (Figure S12). Thus,
glycosidation of 13 to 15 most likely occurs prior to oxidative
decarboxylation of 13.
Figure 5. Ppant ejection mass spectra of adducts (A) 23 and (B) 24 in the
18
presence of
O
2
+ natural abundance H
18
2 2
O + H O (lower trace). (C) Ppant ejection mass spectra of SdmG reaction
2
O (upper trace) or natural abundance
mixture without and (D) with excess 0.1% TFA pre-treatment.
Given that the C-glycosylation products 15 and 16 carry the
same pyrrole moiety as 13, they may also undergo autoxidation
in air to generate 29 and 31, respectively. To trap and
characterize the anticipated oxidative decarboxylation products, a
coupled assay of 12 and 14 with SdmG/SdmA was conducted
under aerobic conditions. When 50 μM 12 was incubated with 0.2
μM SdmG, 10 μM SdmA in the presence of 0.5 mM ribose 5-
To further investigate the mechanism by which 13 undergoes
oxidative decarboxylation, 50 μM 12 was incubated with 1 μM
SdmG in Tris buffer (pH 8.0) under an 18O
atmosphere for 3 h. A
2
mass increase of 2 Da was observed with both 23 and 24
indicating incorporation of a single 18O center from molecular
oxygen to each (Figures 5A and 5B). In contrast, neither adduct
contained 18O when the incubation was conducted with natural
2
phosphate (14), 5 mM MgCl in Tris buffer (pH 8.0), LC-MS
abundance O
2
and 18O-enriched water. The absence of 18
O
analysis revealed the presence of two new SdmD-bound adducts
29 and 30 accompanied by a small amount of 23 and 24 due to
autoxidation of the intermediate 13 (Figures 6C and S13).
Adducts 29 and 30 were not detected when R5P was replaced by
PRPP, which is consistent with the aforementioned results of the
anaerobic assays. Moreover, when SdmB was included in the
coupled reaction assay, products with the correct mass for 26 as
well as showdomycin (1) covalently linked to holo-SdmD (i.e.,
adducts 31 and 32, respectively, Figure S14) could be detected.
To directly test if 16 can be converted to showdomycin via
autoxidation, the SdmA/SdmB coupled reaction filtrate from
anaerobic incubation was exposed to air for 3 h during which time
incorporation from solvent in 24 suggested that 24 might be
derived from the hydrolysis of 23 during HPLC separation and/or
LC-MS analysis rather than during the assay. Indeed, 23 was
found to be stable at pH 8.0 (SdmG assay conditions) but could
be converted to 24 at pH 2.0 (HPLC and LC-MS conditions) as
shown in Figures 5C and 5D. These results agree with the
proposed transformation of 13 to 21. In a separate experiment
where the SdmG reaction was carried out anaerobically and the
reaction filtrate was promptly analyzed by LC-MS, a signal
+
5 7 2 2
consistent with 13 was observed directly (calc. m/z for C H N O
[
M+H]+ 127.0508; found 127.0503) along with the in-source
+
+H]+
decarboxylated fragment (calc. m/z for C
4
H
7
N
2
[M‒CO
2
the correct mass of 26 was detected (calc. m/z for C
9
H
11
N
2
O
5
[M-
-
8
3.0609; found 83.0604) (Figure S8).
The demonstrated reactivity of 13 toward O
H] 227.0668, found 227.0675). When this mixture was acidified
with excess 0.1% formic acid (FA) to pH ~3 for 2 h, LC-MS
analysis revealed the presence of both showdomycin (1) and the
C1' epimer of showdomycin (1'-epi-1) (Figure 6D). Two new
2
suggested that it
is electron-rich and may thus serve as a nucleophile in the
formation of the C-ribosyl linkage catalyzed by SdmA, which is
4
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RESEARCH ARTICLE
peaks (P3 and P4) which have the same exact mass as 1 and 1'- Conclusion
epi-1 were also noted. These species were assigned to be
pyranose isomers of showdomycin (33 and 34) based on their
coelution with the chemically prepared standards. These
observations are consistent with the proposed pathway in Figure
The biosynthetic pathway for showdomycin as encoded by the
sdm gene cluster has now been reconstituted in vitro. The
biosynthetic precursors to showdomycin are L-glutamine (9) and
ribose 5-phosphate (14) with no apparent dependence on PRPP
in contrast to the pyrazole C-nucleosides formycin A (2) and
pyrazofurin (3).[14-16] Assembly is initiated by SdmE catalyzed
cyclization of L-glutamine to yield a 2-amino-1-pyrroline-5-
carboxylate intermediate that is subsequently loaded onto the
phosphopantetheine arm of the peptidyl carrier protein SdmD (9
2
C whereby 16 is a key intermediate en route to the observed
natural product showdomycin (1).
A
→
10 → 11). Oxidation of the resulting thioester-linked pyrroline
is then catalyzed by the flavoenzyme SdmF (11 → 12) before
SdmG mediated hydrolysis to yield the free 2-amino-1H-pyrrole-
5
-carboxylate species 13 as the key intermediate. While it is
unclear why an NRPS system (SdmC and SdmD) participates in
producing 13, it nevertheless is necessary as SdmF is unable to
catalyze the direct oxidation of 10. In contrast, kosinostatin
biosynthesis, which involves homologs of the showdomycin
SdmE, SdmC, SdmD, SdmF and SdmG enzymes, has been
proposed to require further NRPS catalyzed modifications in
addition to oxidation.[22] Thus, while showdomycin and
kosinostatin biosynthetic gene clusters may share a common
evolutionary ancestor, further development may have led to the
observed specificity of SdmF despite other NRPS functions
having been lost.
B
(UV=288 nm)
C
(UV=220 nm)
1
6
holo-SdmD
30
+SdmA & B (R5P)
29
+SdmA (R5P)
1
3
2
4
+SdmA (PRPP)
23
+SdmA (PRPP)
1
5
+
SdmA (R5P)
+SdmA (no R5P)
No Enzyme (R5P)
No Enzyme (R5P)
Retention time (min)
Retention time (min)
D
EIC m/z = 228.05
x105
x 0.02
34
34 standard
3
3
x 0.02
x 0.02
x 0.02
3
3 standard
1
'-epi-1
1
1'-epi-1
standard
Figure 7. Proposed mechanism of glycosidic bond formation catalyzed by
SdmA. Based on sequence alignment with YeiN[17], Lys170 in SdmA is
1
standard
P3
proposed to form a Schiff base with the ring-opened ribose 5-phosphate
P4
+ O
2
/ 3 h
then FA / 2 h
precursor (35) prior to the nucleophilic addition of 13.
+
2
O 3 h
The key step in the showdomycin biosynthetic pathway is
regioselective formation of the C–C linkage between C3 of 13 and
C1′ of ribose 5-phosphate (14) to yield 15, which is unstable in the
presence of molecular oxygen. This reaction is catalyzed by
SdmA. Given the high sequence homology between SdmA and
the pseudouridine monophosphate C-glycosidase YeiN (42%
identity, 59% similarity) as well as their dependence on ribose 5-
phosphate but not PRPP, an analogous C-glycosidation
mechanism can be envisioned as depicted in Figure 7.[
Subsequent dephosphorylation catalyzed by SdmB yields the
showdomycin precursor 16 that is similarly unstable towards
autoxidation. Although showdomycin and 1'-epi-showdomycin
were both detected as autoxidation products of 16, this result
Retention time (min)
Figure 6. (A) Products generated in the reactions catalyzed by SdmA and SdmB.
The nascent enzyme products 15 and 16 were susceptible to oxidative
decarboxylation and covalent modification by holo-SdmD. (B) HPLC analysis of
anaerobic incubations of SdmA and SdmB. (C) HPLC analysis of aerobic
incubations of 12 and SdmA coupled with SdmG. (D) LC-MS analysis of
autoxidation of 16. Extracted ion chromatogram (EIC) traces corresponding to
M - H] signals from 1 and 1'-epi-1 are shown. The unknown P3 and P4 have
the same exact mass as showdomycin and have the same retention time with
standards of pyranose isomer of showdomycin (33 and 34, respectively).
Proposed mechanism for the formation of 1'-epi-1, 33 and 34 is shown in Figure
S15.
17,18]
-
[
5
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cannot be referred to the stereochemistry of 16 or 15 without more
direct evidence. This is because C-nucleosides are known to be
susceptible to epimerization at C1' under acidic conditions.[33-36]
The final tailoring steps that convert 16 to showdomycin are
expected to include oxidation, decarboxylation and deamination
reactions. However, the observed instability of compound 16
indicates that the final steps of showdomycin formation can
proceed via a series of nonenzymatic transformations to afford 1,
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Hence, the C-nucleoside 16 may actually be the true product of
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Keywords: C-nucleoside • maleimide • biosynthesis • C-
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RESEARCH ARTICLE
Entry for the Table of Contents
C-nucleoside biosynthesis: The biosynthetic pathway of the C-nucleoside showdomycin was reconstituted in vitro. Key steps
include cyclization of L-glutamine to yield an oxygen-labile pyrrole intermediate that serves as the substrate for C-glycosidation
catalyzed by SdmA. Autoxidative maturation of the pyrrole nucleobase to the maleimide moiety of showdomycin can then proceed
non-enzymatically.
7
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