Identification of the C-Glycoside Synthases during Biosynthesis of the Pyrazole-C-Nucleosides Formycin and Pyrazofurin

Article abstract of DOI:10.1002/anie.201910356

C-Nucleosides are characterized by a C?C rather than a C?N linkage between the heterocyclic base and the ribofuranose ring. While the biosynthesis of pseudouridine-C-nucleosides has been studied, less is known about the pyrazole-C-nucleosides such as the formycins and pyrazofurin. Herein, genome screening of Streptomyces candidus NRRL 3601 led to the discovery of the pyrazofurin biosynthetic gene cluster pyf. In vitro characterization of gene product PyfQ demonstrated that it is able to catalyze formation of the C-glycoside carboxyhydroxypyrazole ribonucleotide (CHPR) from 4-hydroxy-1H-pyrazole-3,5-dicarboxylic acid and phosphoribosyl pyrophosphate (PRPP). Similarly, ForT, the PyfQ homologue in the formycin pathway, can catalyze the coupling of 4-amino-1H-pyrazole-3,5-dicarboxylic acid and PRPP to form carboxyaminopyrazole ribonucleotide. Finally, PyfP and PyfT are shown to catalyze amidation of CHPR to pyrazofurin 5′-phosphate thereby establishing the latter stages of both pyrazofurin and formycin biosynthesis.

Full text of DOI:10.1002/anie.201910356

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International Edition: DOI: 10.1002/anie.201910356
German Edition: DOI: 10.1002/ange.201910356
Biosynthesis
Identification of the C-Glycoside Synthases during Biosynthesis of the
Pyrazole-C-Nucleosides Formycin and Pyrazofurin
Daan Ren+, Shao-An Wang+, Yeonjin Ko, Yujie Geng, Yasushi Ogasawara, and Hung-wen Liu*
ꢀ
Abstract: C-Nucleosides are characterized by a C C rather
ꢀ
than a C N linkage between the heterocyclic base and the
ribofuranose ring. While the biosynthesis of pseudouridine-C-
nucleosides has been studied, less is known about the pyrazole-
C-nucleosides such as the formycins and pyrazofurin. Herein,
genome screening of Streptomyces candidus NRRL 3601 led to
the discovery of the pyrazofurin biosynthetic gene cluster pyf.
In vitro characterization of gene product PyfQ demonstrated
that it is able to catalyze formation of the C-glycoside
carboxyhydroxypyrazole ribonucleotide (CHPR) from 4-hy-
droxy-1H-pyrazole-3,5-dicarboxylic acid and phosphoribosyl
pyrophosphate (PRPP). Similarly, ForT, the PyfQ homologue
in the formycin pathway, can catalyze the coupling of 4-amino-
1H-pyrazole-3,5-dicarboxylic acid and PRPP to form carbox-
yaminopyrazole ribonucleotide. Finally, PyfP and PyfT are
Figure 1. Representatives of C-nucleoside antibiotics (1–8) and cofor-
mycin (9).
shown to catalyze amidation of CHPR to pyrazofurin 5’-
phosphate thereby establishing the latter stages of both
pyrazofurin and formycin biosynthesis.
T
he C-nucleosides are a noncanonical class of nucleosides in
which the heterocyclic base and the ribofuranosyl core are
ever, how the C-nucleosides are biosynthesized in nature
remains largely unexplored.
Early studies have shown that pseudouridine (1) is
derived from uridine (10) in a reaction catalyzed by pseu-
douridine synthase.[29] The catalytic cycle of this enzyme is
ꢀ
connected through a C C bond rather than the more common
C N linkage that is found in the canonical nucleosides.[1–3] A
ꢀ
wide variety of nucleobases and functional groups are found
in the naturally occurring C-nucleosides including uracil in
pseudouridine (1),[4] malayamycin (2),[5,6] and pseudouridi-
mycin (3),[7,8] the oxazine dione group in minimycin (4),[9,10]
the maleimide unit in showdomycin (5),[11–14] and the pyrazole
moiety in pyrazofurins (6)[15] and formycins (7 and 8)[16,17] (see
Figure 1). Many C-nucleosides found in nature are known to
have antibiotic and antiviral activities mainly due to their
structural resemblance to the N-nucleosides that results in
their interference with primary metabolic processes.[5,7,9,18–27]
Their biological activities have thus inspired the synthesis of
many nonnatural C-nucleosides, some of which have been
demonstrated to be effective therapeutic agents.[2,3,28] How-
ꢀ
initiated by C N bond cleavage to release uracil (11) and an
oxocarbenium ion (12) (or a 1,2-glycal species after C2
deprotonation). Reorientation of 11 in the active site followed
by nucleophilic addition back to 12 can then lead to C-
glycosidic bond formation in 1 as shown in Scheme 1A.[30–32]
PumJ, a pseudouridine synthase equivalent, has been dem-
onstrated to catalyze the same reaction during the biosynthe-
sis of pseudouridimycin (3).[8] The discovery of sdmA, which
encodes a pseudouridine synthase homologue in the show-
domycin (5) gene cluster, likewise suggested a similar mech-
ꢀ
anism of C C bond formation in showdomycin assembly (see
Scheme 1B).[14]
[*] D. Ren,[+] S.-A. Wang,[+] Y. Ko, Y. Geng, Y. Ogasawara,
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
Y. Ogasawara
Current address: Graduate School of Engineering,
Sapporo, Hokkaido 060-8628 (Japan)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201910356.
Scheme 1. Proposed mechanism for pseudouridine synthase. The
cosubstrate 13 in SdmA reaction is likely derived from l-gluta-
mine.[11,12]
.
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In contrast, little is known about the mode of C-glycosidic
bond formation in the biosynthesis of pyrazofurin (6) and the
formycins (7/8). As both carry a pyrazole-derived nucleobase
that is different from those in 1–5, enzymes catalyzing the C-
glycosidation in 6–8 likely involve distinct mechanisms from
that of pseudouridine synthase. The formycin biosynthetic
gene cluster (that is, the for cluster) from Streptomyces
kaniharaensis SF-557 has already been identified.[33] In vitro
experiments showed that carboxyaminopyrazole ribonucleo-
tide (CAPR, 14) is a late intermediate in the formycinA
biosynthetic pathway and its maturation to formycinA 5’-
phosphate (18) is sequentially catalyzed according to Sche-
me 2A by ForC, ForB, ForH, ForA, and ForB together with
by which the pyrazofurin gene cluster might be identified in
a genome-wide search of the producing strain. Towards this
goal, a comparative genomics approach was employed to find
the responsible enzymes and investigate the mechanisms of
ꢀ
C C coupling in 6–8. Reported herein is the discovery of
novel enzyme-catalyzed C-glycosidation reactions that under-
lie biosynthesis of the pyrazole group in biological systems.
The genome of Streptomyces candidus NRRL 3601 was
sequenced (9.2 Mbp with 301 contigs) (N50: 152,140 bp), and
the genes pyfG, pyfH, and pyfI, which are homologous to forJ,
forK, and forL, were found clustered on the k141_289 contig.
This cluster, designated as the pyf cluster, spans a circa 25 kbp
region between two ABC transporters which are the likely
boundary of the pyf cluster (see Figure 2). BLAST (basic local
Figure 2. The pyrazofurin (pyf) biosynthetic gene cluster from S.
candidus (on contig k141_289) in comparison with the formycin (for)
gene cluster from S. kaniharaensis. The coformycin biosynthetic genes
(cof genes) are found adjacent to the main clusters in both the pyf and
for clusters.
alignment search tool) comparison of the pyf and for clusters
not only revealed the conservation of the putative hydrazine-
assembly genes (forJ/forK/forL versus pyfG/pyfH/pyfI) in
each cluster, but also the presence of forC and forB
homologues, denoted here as pyfP and pyfT, respectively.
These findings are consistent with the pyf cluster being
responsible for pyrazofurin biosynthesis and suggest that
formycins and pyrazofurin may share the same pyrazole
forming machinery. In addition, carboxyhydroxypyrazole
ribonucleotide (CHPR, 19) was considered to be a likely
intermediate of pyrazofurin biosynthesis and specifically
a substrate for sequential reactions catalyzed by the pyfP
and pyfT gene products as shown in Scheme 2A.
Also noted was the absence of forHAFI homologs in the
pyf cluster (see dashed box in Figure 2). This is consistent with
the observation that ForH and ForA are required only for the
construction of the second heterocycle in formycins, which is
absent in pyrazofurin (see Scheme 2A).[33,34] Furthermore, the
coformycin (9) biosynthetic genes (the cof cluster) were
found to be adjacent to both the for and pyf main clusters.[36]
This flanking arrangement is not surprising, because formy-
cins (7/8) are produced synergistically with coformycin (9),
which is an inhibitor of adenosine deaminase (ADA) and can
therefore prevent formycinA (7) deactivation by ADA.[16,37]
Similarly, amide bond hydrolysis of pyrazofurin (23!19) was
also observed when pyrazofurin (6) was incubated with ADA
(type X) (see Figure S2 in the Supporting Information). This
Scheme 2. A Conversion of CAPR (14) to formycinA 5’-phospahte (18)
in the formycin biosynthetic pathway and the intermediacy of CHPR
(19) in pyrazofurin pathway. B Formation of hydrazino acetic acid (20)
in the biosynthesis of s56-p1 (21).[35] C Possible involvement of ForJ/K/
L and PyfG/H/I in hydrazine assembly in formycin and pyrazofurin
biosynthesis, respectively.
PurH, which originates in primary metabolism.[33,34] Impor-
tantly, the forJ, forK, and forL genes in the for cluster were
noted to be homologous to spb38, spb39, and spb40 in the
gene cluster of s56-p1 (21).[35] Enzymes encoded by the latter
set of genes have been shown to catalyze the transformation
of lysine and glycine to give hydrazino acetic acid (20)
enroute to s56-p1 (Scheme 2B).[35] Hence, hydrazine forma-
tion may also be involved in the construction of the pyrazole
group of formycins[33] and perhaps pyrazofurin as well
(Scheme 2C). If correct, this hypothesis suggested a criterion
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Scheme 3. A Reaction catalyzed by b-ribofuranosylaminobenzene 5’-
phosphate synthase. B and C Reactions catalyzed by ForT and PyfQ
using PRPP (24) and different nitrogen-containing compounds as
substrates.
bioinformatic analysis collectively suggested that the pyf
cluster encodes the enzymes of pyrazofurin biosynthesis.
To test the hypothesis that the pyf cluster is indeed
responsible for pyrazofurin biosynthesis, selected pyf gene
products were expressed and characterized in vitro. In
particular, PyfQ as well as ForT from the for cluster are
annotated as encoding a b-ribofuranosylaminobenzene 5’-
phosphate (b-RFA-P) synthase. b-RFA-P synthase has been
demonstrated to catalyze formation of the C-glycoside 27
from phosphoribosyl pyrophosphate (PRPP, 24) and p-
aminobenzoic acid (pABA, 25) during biosynthesis of meth-
anopterin (Scheme 3A).[38–41] Furthermore, this reaction is
believed to involve electrophilic aromatic substitution (EAS)
of pABA (25) with the oxocarbenium ion (12) from PRPP
(24, Scheme 3A).[38,39] Therefore, it was proposed that an
analogous substitution reaction also occurs between PRPP
and an aromatic nucleobase in the catalytic cycles of ForTand
PyfQ such that these two enzymes are responsible for C-
glycosidic bond formation in formycin[33] and pyrazofurin,
respectively.
In order to investigate these hypotheses, the putative six-
membered pyridazinone[33] intermediate 22 was incubated
with ForT and PyfQ, but no C-glycosidation product was
observed (Scheme 3B and Figure S3 in the Supporting
Information). However, when 4-amino-1H-pyrazole-3-car-
boxylic acid (APA, 29) was prepared (see Supporting
Information) and incubated with PRPP (24) and ForT for
36 h, two new products were detected (see Figure 3B,
trace 4). These products were identified as aminopyrazole
ribonucleotide (APR, 30) and CAPR (14) based on MS
analysis (see Supporting Information) and HPLC coelution
with the synthetic standards. Although the yield was poor
(< 5%), these results demonstrated that ForT catalyzes a C-
glycosidation reaction. The production of both APR and
CAPR suggested competing electrophilic addition of the
putative oxocarbenium intermediate 12 at C3 versus C5 of 29
to yield APR (30) versus CAPR (14), respectively (see
Figure 3. A ForT/PyfQ reactions using APA (29) as substrate. B HPLC
analysis of ForT/PyfQ reactions with APA (29).
Figure 3A, routes a vs. b). While ForT appeared to promote
addition at both C3 and C5, the same experiment with PyfQ
demonstrate primarily decarboxylative EAS (route a) at C3
to yield APR (30) with essentially no addition at C5 (see
Figure 3B, trace 5). Furthermore, APA (29) appeared to be
a much better substrate for PyfQ compared to ForT showing
significantly greater turnover during the incubation time
period.
The poor activity of ForTon APA indicated that APA may
not be the correct substrate for ForT. The observed decar-
boxylation of APA (29) to APR (30) also implied that a C3
carboxylation reaction is required to convert APR to CAPR
(14) or a derivative thereof in the biosynthesis of both
formycins and pyrazofurin (see Figure 3A). However, no
genes in either cluster were annotated to encode a carbox-
ylase. Furthermore, neither PurE nor PurK, which catalyze an
analogous carboxylation reaction in de novo purine biosyn-
thesis,[42,43] led to CAPR (14) formation when incubated with
APR (30) (see Figure S4, S5 in the Supporting Information).
Moreover, prior labeling experiments have indicated that
both the C6 of formycinA and the amide carbon of
pyrazofurin originate from C1 of glutamate rather than
bicarbonate.[44] Taken together, APA (29) is unlikely to be
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a biosynthetic intermediate in the formycin and pyrazofurin
pathways.
Figure 5A). When purified PyfP was incubated with 19, l-
Asp, and ATP, a single product was produced (see Figure 5B,
trace 3) and displayed a mass consistent with succinyl-
If ForT and PyfQ are indeed the C-glycosidases of
formycin and pyrazofurin biosynthesis, then the carboxylate
group at C3 carbon of their pyrazole substrates should be
retained. Therefore, 4-amino-1H-pyrazole-3,5-dicarboxylic
acid (APDA, 31) and 4-hydroxy-1H-pyrazole-3,5-dicarbox-
ylic acid (HPDA, 32) were prepared (see Supporting Infor-
mation) and tested for activity with ForT and PyfQ (see
Figure 4A). Indeed, incubation of APDA (31) with PRPP
Figure 5. A Reactions of CHPR (19) with PyfP and PyfT. B HPLC
analysis of the PyfP and PyfT reactions.
hydroxypyrazole carboxamide ribonucleotide (SHPCAR,
34) (calcd m/z for C13H17N3O13P [MꢀH]ꢀ 454.0499; found
454.0529). Furthermore, addition of purified PyfT, a putative
adenylsuccinate lyase, to PyfP previously incubated with 19,
l-Asp, and ATP, led to consumption of 34 and formation
a new compound (Figure 5B, trace 4) having the expected
mass 338.0384 of pyrazofurin 5’-phosphate (23, calcd m/z for
C9H13N3O9P [MꢀH]ꢀ 338.0389). Confirmation of this assign-
ment was obtained when the PyfQ/PyfP/PyfT product from 32
was isolated, treated with calf intestinal phosphatase and
found to coelute with a standard of pyrazofurin (6) on HPLC
(see Figure S7 in the Supporting Information). It was also
found that ForC and PurC, which are homologues of PyfP in
the formycin and de novo purine pathways, also catalyzed the
conversion of 19 to 34 (see Figure S8 in the Supporting
Information).
Figure 4. ForT/PyfQ reactions with A APDA (31). B With HPDA (32).
C With PDA (33). D HPLC analysis of ForT/PyfQ reactions with APDA
(31). E HPLC analysis of ForT/PyfQ reactions with HPDA (32).
(24) in the presence of either ForTor PyfQ led to quantitative
conversion of APDA to CAPR (14) within 1 hour (see
Figure 4D). Similarly, PyfQ catalyzes complete conversion of
HPDA (32) and PRPP (24) to CHPR (19) within 1 hour of
incubation (see Figure 4E); however, 32 was not found to be
a substrate for ForT. These results strongly support correct
assignment of the clusters and indicate that ForT exhibits
greater substrate specificity compared to PyfQ. Moreover, no
reaction was observed with either ForT or PyfQ when 1H-
pyrazole-3,5-dicarboxylic acid (PDA, 33) was supplied as the
substrate (Figure 4C and Figure S6 in the Supporting Infor-
mation). This implies that an electron donating group is
required to increase the nucleophilicity of the heterocycle and
is consistent with a C-glycosidation mechanism that involves
EAS.
In summary, PyfQPTencoded in the pyf cluster[45] are able
to catalyze the conversion of HPDA (32), PRPP (24), and l-
aspartate to pyrazofurin 5’-phosphate (23). Similarly, ForT
catalyzes the coupling of APDA (31) and PRPP (24) to
produce CAPR (14), which is subsequently converted to
formycinA 5’-phosphate (18) in reactions catalyzed by
ForABCH and PurH.[33,34] In particular, PyfQ as well as
ForTare C-glycoside synthases[46] and are likely characterized
by catalytic cycles that involve EAS reactions. These obser-
vations indicate that the for and pyf clusters are indeed
responsible for formycin and pyrazofurin biosynthesis and
thus establish the latter stages of both biosynthetic pathways.
Nevertheless, the early stages of these pathways involving the
assembly of the pyrazole rings in APDA (31) and HPDA (32)
remain to be determined and may involve a hydrazine-
generating process (see Scheme S3 in the Supporting Infor-
mation) analogous to that found in the s56-p1 biosynthetic
pathway.[35]
To further investigate the pyf gene cluster, the CHPR (19)
product of the PyfQ-catalyzed reaction with HPDA (32) was
investigated as a substrate for the pyfP-encoded gene
product. PyfP is annotated as a phosphoribosylaminoimida-
zole-succino-carboxamide (SAICAR) synthetase and thus
predicted to catalyze amidation of 19 with aspartate (see
4
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Acknowledgements
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Manuscript received: August 13, 2019
Accepted manuscript online: September 13, 2019
Version of record online: && &&, &&&&
Angew. Chem. Int. Ed. 2019, 58, 1 – 6
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Biosynthesis
Enzymatic C-glycoside bond formation:
The C-glycoside synthases encoded in the
formycin and pyrazofurin biosynthetic
gene clusters are identified and com-
pared by genomic analysis of the pro-
ducing strains. Both enzymes catalyze
decarboxylative C-glycoside bond forma-
tion between phosphoribosyl pyrophos-
phate and a pyrazole nucleobase in
reactions likely to involve electrophilic
aromatic substitution.
D. Ren, S.-A. Wang, Y. Ko, Y. Geng,
Y. Ogasawara, H.-w. Liu*
&&&& — &&&&
Identification of the C-Glycoside
Synthases during Biosynthesis of the
Pyrazole-C-Nucleosides Formycin and
Pyrazofurin
6
www.angewandte.org
ꢀ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2019, 58, 1 – 6
These are not the final page numbers!