The Amipurimycin and Miharamycin Biosynthetic Gene Clusters: Unraveling the Origins of 2-Aminopurinyl Peptidyl Nucleoside Antibiotics

Article abstract of DOI:10.1021/jacs.9b03021

Peptidyl nucleoside antibiotics (PNAs) are a diverse class of natural products with promising biomedical activities. These compounds have tripartite structures composed of a core saccharide, a nucleobase, and one or more amino acids. In particular, amipurimycin and the miharamycins are novel 2-aminopurinyl PNAs with complex nine-carbon core saccharides and include the unusual amino acids (-)-cispentacin and N⁵-hydroxyarginine, respectively. Despite their interesting structures and properties, these PNAs have heretofore eluded biochemical scrutiny. Herein is reported the discovery and initial characterization of the miharamycin gene cluster in Streptomyces miharaensis (mhr) and the amipurimycin gene cluster (amc) in Streptomyces novoguineensis and Streptomyces sp. SN-C1. The gene clusters were identified using a comparative genomics approach, and heterologous expression of the amc cluster as well as gene interruption experiments in the mhr cluster support their role in the biosynthesis of amipurimycin and the miharamycins, respectively. The mhr and amc biosynthetic gene clusters characterized encode enzymes typical of polyketide biosynthesis instead of enzymes commonly associated with PNA biosynthesis, which, along with labeled precursor feeding studies, implies that the core saccharides found in the miharamycins and amipurimycin are partially assembled as polyketides rather than derived solely from carbohydrates. Furthermore, in vitro analysis of Mhr20 and Amc18 established their roles as ATP-grasp ligases involved in the attachment of the pendant amino acids found in these PNAs, and Mhr24 was found to be an unusual hydroxylase involved in the biosynthesis of N⁵-hydroxyarginine. Finally, analysis of the amc cluster and feeding studies also led to the proposal of a biosynthetic pathway for (-)-cispentacin.

Full text of DOI:10.1021/jacs.9b03021

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Article
The Amipurimycin and Miharamycin Biosynthetic Gene Clusters:
Unraveling the Origins of 2-Aminopurinyl Peptidyl Nucleoside Antibiotics
Anthony J Romo, Taro Shiraishi, Hideo Ikeuchi, Geng-Min Lin, Yujie Geng, Yu-Hsuan Lee, Priscilla H. Liem,
Tianlu Ma, Yasushi Ogasawara, Kazuo Shin-ya, Makoto Nishiyama, Tomohisa Kuzuyama, and Hung-wen Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b03021 • Publication Date (Web): 31 May 2019
Downloaded from http://pubs.acs.org on May 31, 2019
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The Amipurimycin and Miharamycin Biosynthetic Gene Clus-
ters: Unraveling the Origins of 2-Aminopurinyl Peptidyl Nucle-
oside Antibiotics
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a,1,‖
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Anthony J. Romo, Taro Shiraishi, Hideo Ikeuchi, Geng-Min Lin, Yujie Geng, Yu-Hsuan Lee, Priscilla
e
e,3
a,4
c,f
c,g
b,g,*
H. Liem, Tianlu Ma, Yasushi Ogasawara, Kazuo Shin-ya, Makoto Nishiyama, Tomohisa Kuzuyama,
a,d,e,*
and Hung-wen Liu,
a
Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712,
b
USA; Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan;
c
d
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; Department of Chemis-
e
f
try; and Department of Biochemistry, The University of Texas at Austin, Austin, TX 78712, USA; National Institute of Advanced
Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan; Collaborative Research Institute for Innovative
g
Microbiology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
KEYWORDS: Peptidyl nucleoside antibiotics, natural product biosynthesis, Streptomyces, nucleoside, ATP-grasp ligase
ABSTRACT: Peptidyl nucleoside antibiotics (PNAs) are a diverse class of natural products with promising biomedical activities. These com-
pounds have tripartite structures composed of a core saccharide, a nucleobase, and one or more amino acids. In particular, amipurimycin and the
miharamycins are novel 2-aminopurinyl PNAs with complex nine-carbon core saccharides and include the unusual amino acids (–)-cispentacin
5
and N -hydroxyarginine, respectively. Despite their interesting structures and properties, these PNAs have heretofore eluded biochemical scrutiny.
Herein is reported the discovery and initial characterization of the miharamycin gene cluster in Streptomyces miharaensis (mhr) and the amipurimy-
cin gene cluster (amc) in Streptomyces novoguineensis and Streptomyces sp. SN-C1. The gene clusters were identified using a comparative genomics
approach, and heterologous expression of the amc cluster as well as gene interruption experiments in the mhr cluster support their role in the bio-
synthesis of amipurimycin and the miharamycins, respectively. The mhr and amc biosynthetic gene clusters characterized encode enzymes typical
of polyketide biosynthesis instead of enzymes commonly associated with PNA biosynthesis, which along with labeled precursor feeding studies,
implies that the core saccharides found in the miharamycins and amipurimycin are partially assembled as polyketides rather than derived solely
from carbohydrates. Furthermore, in vitro analysis of Mhr20 and Amc18 established their roles as ATP-grasp ligases involved in the attachment of
5
the pendant amino acids found in these PNAs, and Mhr24 was found to be an unusual hydroxylase involved in the biosynthesis of N -
hydroxyarginine. Finally, analysis of the amc cluster and feeding studies also led to the proposal of a biosynthetic pathway for (–)-cispentacin.
Introduction
inherent classification scheme, it is likely an oversimplification of
the biosynthetic space occupied by this class of natural products
given the paucity of sequenced PNA biosynthetic gene clusters
Peptidyl nucleoside antibiotics (PNAs) are composed of a nu-
cleobase, a core saccharide, and one or more appended amino ac-
ids. They comprise an underexploited, structurally heterogeneous
family of natural products that has begun to garner more attention
2
,3
compared to the number and diversity of known PNA structures.
Thus, investigation of how PNAs are constructed holds promise for
both the development of useful therapeutics and the enrichment of
our understanding of natural product biosynthesis.
5
1
as a potential new source of antimicrobials. However, the lack of
structural conservation between PNAs and other more widely stud-
ied classes of natural products presents a formidable challenge to
their biosynthetic study. Efforts to determine PNA biosynthetic
pathways are further complicated by their use of extensively modi-
fied nucleobases, higher-carbon sugars, and non-proteinogenic
N "hydroxylarginine/arginine
(")"cispentacin
2,3
amino acids in the assembly. Several PNA biosynthetic gene clus-
ters have been sequenced to date, and subsequent characterization
has revealed that the core saccharides of these PNAs originate from
only one of four different precursors: a nucleoside diphosphate
(NDP)-activated deoxy sugar, uridine diphosphate (UDP)-
activated glucuronic acid, an intact nucleoside from primary me-
perhydrofuropyran
saccharide:core
open"branched:chain
saccharide core
2"aminopurine
2"aminopurine
Miharamycin A,:R:=:OH:(
Miharamycin B,:R=:H:(
1
)
Amipyurimycin (
3)
2
)
Figure 1. Comparison of the structural features of miharamycins
1 and 2) and amipurimycin (3).
4
,5
(
tabolism, or octosyl acid. Despite the potential utility of such an
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The miharamycins (1, 2) and amipurimycin (3) are PNAs pro-
Additional evidence for the assignment of the mhr and amc clus-
ters was obtained from an analysis of their putative regulatory ele-
ments. The ORFs amc0 and amc16 are annotated as encoding pro-
teins that contain a SARP-responsive bacterial transcriptional acti-
vator domain, which indicated these proteins might increase ex-
pression of the amc biosynthetic gene cluster. With this in mind,
amc0 and amc16 were individually cloned and introduced into S.
sp. SN-C1. Fermentation of the transformants showed significantly
increased amipurimycin production, with up to 30 times the basal
production level observed when amc0 or amc16 were individually
overexpressed (Figures 2C and 2D). Altogether, the heterologous
expression of the amc cluster and subsequent observation of the
potent effect of the amc0 and amc16 gene products on amipurimy-
cin overproduction, as well as the inactivating gene deletions per-
formed in S. miharaensis strongly implicate a direct role of the amc
and mhr clusters in the biosynthesis of amipurimycin and the miha-
ramycins, respectively.
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duced by the actinomycetes Streptomyces miharaensis and Strepto-
myces novoguineensis, respectively, exhibiting diverse antifungal,
antibacterial, and antiviral activities, the mechanisms of which are
6
-9
currently undetermined. The miharamycins and amipurimycin
are each structurally composed of the rare nucleobase 2-
aminopurine, a structurally complex core saccharide, and a single
2
3
1
0,11
amino acid each. Miharamycin A and amipurimycin are deco-
5
rated by the non-proteinogenic amino acids N -hydroxyarginine
and (1R,2S)-2-aminocyclopentane-1-carboxylic acid (also known
as (–)-cispentacin), respectively, which themselves display mild
0
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antibiotic properties.
Besides their pendant amino acids, the
miharamycins are distinguished from amipurimycin by their bicy-
clic perhydrofuropyran saccharide cores (Figure 1). The biosyn-
thetic gene clusters and pathways for amipurimycin and the miha-
ramycins have not been reported to date. Interestingly, while the
16
total synthesis of amipurimycin was accomplished only recently,
some 40 years after the initial discovery of this PNA, a successful
17,18
total synthesis of a miharamycin has yet to be reported.
Herein, we identify and characterize the biosynthetic gene clus-
ters of the miharamycins and amipurimycin from S. miharaensis and
S. novoguineensis, respectively. The presence of genes encoding
characteristic polyketide biosynthetic enzymes along with labeled
precursor feeding studies imply that the core saccharides of these
PNAs are likely partially polyketide-derived. This finding suggests a
new paradigm for the biological production of unusual sugars, and
allows us to propose a biosynthetic pathway for amipurimycin.
Moreover, insight into the biosynthesis and ATP-grasp ligase-
mediated attachment of the pendant amino acids decorating ami-
purimycin and the miharamycins is also provided.
Results and Discussion
Comparative genomics approach to gene cluster discovery. The S.
novoguineensis and S. miharaensis genomes were sequenced and
then annotated utilizing Rapid Annotation Using Subsystem Tech-
Figure 2. (A) Extracted ion current chromatogram comparing culture
extracts from S. sp. SN-C1 (positive control) and S. albus G153 trans-
formed with empty pKU503D vector (negative control) or
pKU503ampr1, demonstrating heterologous expression of the amc
cluster in S. albus G153; (B) HPLC chromatograms (λ= 305 nm)
demonstrating loss of production of miharamycins in S. miharaensis as
a result of selected gene deletions; (C) over-production of amipurimy-
cin in S. sp. SN-C1 when amc0 is supplemented; (D) over-production
of amipurimycin in S. sp. SN-C1 when amc16 is supplemented.
1
9-21
nology (RAST).
Scanning for shared gene clusters using an-
2
2
tiSMASH resulted in the identification of a single unique gene
cluster shared between both strains at an approximately 30 kbp
locus in each genome. The shared putative clusters contained 30
open reading frames (ORFs) in the 32.5 kbp conserved region
found in S. novoguineensis (the amc cluster) and 27 ORFs in the
2
9.2 kbp conserved region found in S. miharaenesis (the mhr clus-
ter). A duplicate, identical amc cluster was also found in Streptomy-
ces sp. SN-C1 (see Supporting Information). The gene clusters are
shown schematically in Figure S-3.3, and ORFs from both putative
gene clusters are listed in Tables S-3.3a and S-3.3b along with their
predicted products. Gene cluster boundaries were approximated as
described in the Supporting Information (Section S-3.4).
Gene cluster validation. A bacterial artificial chromosome (BAC)
containing the amc cluster was isolated (see Section S-4.1) in order
to further characterize this gene cluster. The BAC clone,
pKU503ampr1, was transformed into the non-producing host
Streptomyces albus G153 via protoplast transformation, converting
it to an amipurimycin-producing strain (see Figure 2A). While the
same approach was not pursued with the mhr gene cluster, double
crossover-mediated deletion of mhr5, mhr7, or mhr17 in S. mi-
haraensis completely ablated production of the miharamycins (see
Figure 2B).
Proposed biosynthetic pathway based on feeding studies and gene
cluster analyses. The biosyntheses of all of the PNAs discovered to
date involve the incorporation of an intact sugar precursor mole-
cule into the final structure, e.g., glucuronic acid for the cytosyl
PNAs, frequently via the nucleoside diphosphate activated form of
these sugars. Towards providing the first insight into the biosynthe-
sis of amipurimycin and the miharamycins, we chose to focus on
13
amipurimycin and administered uniformly C-labeled glucose
13
([U- C
6
]glucose, 4) to the culture broth of S. sp.SN-C1/
pSE101amc0. The labeled amipurimycin (5) was isolated and
analyzed by C NMR. The C NMR spectrum revealed efficient
C incorporation of the core saccharide (Fig. S-5.1). Remarkably,
13
13
13
carbon–carbon coupling analyses using 2D-INADEQUATE (in-
credible natural-abundance double-quantum transfer experiment)
spectroscopy did not support incorporation of an intact sugar mol-
ecule (Fig. S-5.2–S-5.4). Instead, the observed coupling patterns
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showed the core saccharide to be composed of three distinct frag- thiolation of the atypical PKS extender unit hydroxymalonate (7)
2
4a
1
2
3
4
5
6
7
8
9
ments, C1'–C2', C3'–C4'–C5'–C6', and C8'–C9', with C7' labeled
but uncoupled to any other carbon atom (see 5 in Fig. 3). Unlike
the previously characterized PNA biosynthetic pathways, this un-
expected pattern does not support the intermediacy of nucleoside
diphosphate-activated sugars and suggests the biosyntheses of ami-
purimycin and the miharamycins deviate substantially from other
PNA biosynthetic pathways.
in polyketide biosynthetic pathways.
Given the simple composition of the Amc8 enzyme, this PKS en-
zyme would be expected to be capable of only one round of chain
extension. In consideration of the hydroxymalonate cassette found
in the amc cluster, as well as the predicted preference of the AT
domain in Amc8 for hydroxymalonate via the ClusterCAD serv-
24b
er, it can be reasoned that Amc8 uses a two-carbon fragment
glucose
from ACP-linked hydroxymalonate (derived from 7) in its exten-
sion reaction (8 ® 9). We, thus, predict the hydroxymalonate-
derived unit results in the labeled C1'–C2' fragment observed in the
structure of amipurimycin. However, this only accounts for two of
the nine carbons composing the core saccharide of amipurimycin.
As the "acceptor" of the two-carbon extender unit (i.e., the PKS
starter unit) is likely a linear five-carbon fragment (such as 8 shown
in Figure 3), addition of two more carbons is needed to complete
the assembly of the branched nine-carbon core. Such two-carbon
extension could be a post-PKS event or may occur while the carbon
chain is still attached to Amc8.
O
OH
HO
O
O
O
H
O
OH
OH
OH
O
OH
OH
O
O
HO
HO
HO
HO
HO
HO
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HO
HO
OH
OH
OH
OH
OH
OH
OH
+
HO
OPO3=
OPO3=
OPO
glyceraldehyde
-phosphate
=
3
OPO
3
=
OPO
3
=
OPO
3
=
OPO =
3
ribose 5-
phosphate
xylulose 5-
phosphate
3
fructose 6-
phosphate
glucose 6- 6-phospho-glucono-
phosphate
6-phospho-
gluconate
transketolase
δ-lactone
trans-
ketolase
CO
2
OH
HO
O
OH
OH
O
O
O
O
trans- HO
HO
HO
O
OH
OH
OH
O
aldolase
OH
+
OH
OH
+
OH
HO
HO
HO
HO
OH
OPO =
OH
OPO3=
OPO3=
OH
erythrose 4-
phosphate
OH
xylose
3
OPO3=
OPO =
glyceraldehyde
3-phosphate
OPO3
=
The C8'–C9' fragment which was labeled in one piece in the
3
xylulose 5-
phosphate
ribulose 5-
phosphate
sedoheptulose
7-phosphate
fructose 6-
phosphate
feeding experiments performed in S. sp. SN-C1/pSE101amc0 fed
1
3
with [U- C ]glucose (4) constitutes the two-carbon branch in the
6
Weimberg pathway (oxidative xylose degradation)
structure of amipurimycin. Recent investigations have demonstrat-
ed PKSs can effect branching through the use of specialized PKS
Amc8
Amc8
Amc8
OH
O
O
KS AT ACP
KS AT ACP
KS AT ACP
O
25
S
S
S
SH
O
S
modules or a conserved set of "branching" enzymes. However,
Amc8 and the amc cluster lack these features. Instead, the amc clus-
ter encodes a two-component transketolase, Amc3a/3b, which, by
Amc7
O
O
O
O
O
OH
O
OH
OH
OH
α-ketoglutarate (
HO
O
HO
HO
6
)
O
O
O
HO OH
26
analogy to the biosynthesis of branched saccharides, would be a
U-13
8
O
[
C
6
]glucose
(
4
)
OH Amc10
OH
Amc10
Amc10
O
O
good candidate to install the C8'–C9' two-carbon fragment (12 ®
13), perhaps originating from ribulose- or xylulose-5-phosphate
(see Figure 3).
ACP
H O
OH
ACP
ACP
2
9
OPO3=
S
O
O
S
S
O
Amc12 O
Amc9 O
Amc11
O
HO
OH
OH
OH
OH
OH
OH
O
OH
HO
OPO3=
O
?
In the course of analyzing the amc and mhr clusters, the discov-
ery of several more homologous gene clusters allowed us to predict
conserved characteristics of ostensibly 2-aminopurinyl (i.e., amipu-
rimycinoid) PNAs (see section S-7). The newly identified amipu-
rimycinoid clusters do not all contain a homolog of Amc3a/3b, but
those that do possess a homologous transketolase also encode an
oxidoreductase homologous to Amc14, which could serve to cata-
lyze reduction of the C8' carbonyl of the Amc3a/3b-installed gly-
coaldehyde, thereby affording the C8' alcohol (as shown in 13)
found in the structure of amipurimycin.
If branching at C3' can be seen as a "tailoring" reaction, then the
true Amc8 starter unit must be composed of the remaining five
carbons, i.e., C3', C4', C5', C6' and C7'. As described above, C3'
through C6' were coupled, while C7' was labeled but never coupled
— a "4+1" labeling pattern. One compelling explanation for this
O
Amc2
2
-hydroxy-malonate
1
,3-bisphospho-
glycerate
HO
(
7
)
HO
guanosine
monophosphate
H
2
N
O
H2
N
Amc1
O
O
O
Amc6
N
N
N
HO OH
Amc15
N
N
O
O
HO
O
=
O3PO
N
NH
2
O
N
H
N
NH2
11
10
HO
OH
2-aminopurine (15
)
Amc4
O
HO
14
HO
HO
7
'
6'
HN
4'
O
N
N
H
2
N
O
N
N
Amc3a/b H2
N
N
N
NH
2
N
Amc18
N
O
O
O
N
HO 3
N
NH2
HO
N
NH2
O
'
1'
Amc14
HO
N
NH
2
HO
HO
OH
HO 8'
9'
HO
HO
1
3
12
OH
labeled amipurimycin (
OH
5
)
O
(
-)-cispentacin (16)
H
2
N
Figure 3. Proposed biosynthetic pathway for amipurimycin based
on gene cluster analysis and results of feeding experiments. The
labeled α-ketoglutarate is predicted to arise from [U- C
glucose via pentose phosphate interconversions and the Weimberg
pathway, while the two-carbon fragment (C8'-C9', green) is pre-
dicted to originate from a ketose-5-phosphate, as described in the
text.
13
6
]-labeled
distinctively labeled C starter unit is that it originates from the
5
transformation of glucose to ribulose to a-ketoglutarate via pentose
phosphate interconversions and the Weimberg pathway (Figure
2
7,28
3).
Thus, while there are few reports exploring the linkage be-
tween primary (e.g., from the Weimberg pathway) and secondary
metabolism in Streptomyces, it is plausible that the starter unit
could be a-ketoglutarate (6, Figure 3). Overall, Amc8 is believed to
catalyze the addition of two-carbon atoms derived from hydroxy-
malonate (7) to a-ketoglutarate to yield a seven-carbon chain (8 ®
Upon closer analysis of the contents of the amc gene cluster, we
found it to encode a mono-modular minimal type I polyketide
synthase (PKS) composed of one ketosynthase (KS), one acyl-
transferase (AT) and one thiolation (T) domain (Amc8) together
with a separate ORF encoding a putative, unusual adenyla-
tion/thiolation-type loading module (Amc7). In addition, the amc
cluster possesses a cassette (amc9–12) encoding four conserved
enzymes (Amc9–12) implicated in the biosynthesis and ACP-
9
), whose cyclization would generate the pyran portion of the ami-
purimycin core saccharide (10). Though its origin is not obvious,
an alternative pathway starting from 2-keto-3-hydroxy-glutarate
could also be possible (see Figure S-5.5). Then, Amc3a/3b catalyz-
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Page 4 of 9
es the addition of two more carbon atoms to complete the nine-
carbon skeleton of amipurimycin (12 ® 13).
that produced amipurimycinine (19, i.e., the nucleoside scaffold of
amipurimycin) as a putative substrate for Amc18. Structural and
spectroscopic characterization of both miharamycinine and amipu-
rimycinine is provided in the Supporting Information (Section S-
6.2 and Figures S-6.2.3–S.6.2.8).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Condensation with 2-aminopurine (15), transamination by
Amc2, and attachment of (–)-cispentacin (16) are three further
PKS tailoring steps (Fig. 3), but the sequence of these events is not
apparent. It remains to be seen whether the same labeling patterns
are observed in the miharamycins. Nevertheless, given the strong
conservation of content between the amc and mhr clusters, the
proposed biosynthetic pathway is expected to be operative in the
assembly of the miharamycins as well. Further work to explore
these biosynthetic pathways is in progress.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Following construction, off-loading, and cyclization of the nas-
cent heptosyl core (10), it may then be converted to the nucleoside
moiety by introduction of the 2-aminopurine nucleobase (15). The
first step is likely reduction of the lactone (10) at the "anomeric"
position catalyzed by the conserved myo-inositol 2-dehydrogenase/
oxidoreductase Amc1. The amc cluster also contains a cistronic
"glycosylation cassette" (e.g., amc4–6) that possess a gene annotat-
ed as encoding an O-kinase, amc5. The co-localization of these
genes suggests that coupling of 11 with 15 may be initiated by C1
phosphorylation followed by substitution with 2-aminopurine. The
latter reaction may be catalyzed by the product of amc4, a predicted
cytosylglucuronic acid (CGA) synthase which mediates the con-
Figure 4. Overview of in vitro assays performed using miharamy-
cinine (17) as the amino acid acceptor; HPLC chromatogram (λ=
305 nm) of assays, as well as mass spectra of the products of the
2
9
assays are shown.
densation between UDP-glucuronic acid and cytosine in the bio-
synthesis of UDP-glucuronic acid-derived PNAs, including blasti-
3
0
Miharamycinine (17) was consumed in the presence of Mhr20
only when co-incubated with both L-arginine and ATP (Figure 4).
The identity of the product was confirmed to be miharamycin B
cidin S, arginomycin, and gougerotin.
The origin of the 2-aminopurine nucleobase itself (15) is un-
clear; however, it may involve the activities of the annotated flavo-
protein Amc6 as well as Amc15, a homolog of the cytokinin matu-
rating phosphoribohydrolase LONELY GUY-1 (LOG-1) from
(
2) by co-elution with the standard compound and high-resolution
mass spectrometry. Similarly, amipurimycin (3) was produced
when Amc18 was incubated with (–)-cispentacin, amipurimycinine
(19), and ATP (Figure 5). The results suggest that Mhr20 and
Amc18 are indeed the ATP-dependent ligases responsible for the
attachment of L-arginine and (–)-cispentacin to their respective
core nucleosides and that amino acid ligation likely occurs late in
the biosynthetic pathways of these PNAs. Moreover, Amc18 could
catalyze the ligation of (–)-cispentacin to miharamycinine (Figure
3
1
Arabidopsis thaliana. Although mechanistically distinct from nu-
cleoside 2′-deoxyribosyltransferases such as BlsM from the blasti-
3
2
cidin S biosynthetic pathway, the LOG-1 homolog Amc15 could
conceivably accomplish the same end, namely, the release of a nu-
cleobase from a donor nucleotide (14 ® 15). Taken together, the
coexistence of a phosphoribohydrolase and CGA synthase homo-
log in the amc cluster implies amipurimycin is derived from glyco-
sylation of a nucleobase (e.g., 15) with a sugar (e.g., 11) and not via
modification of a preformed nucleoside or nucleotide as is ob-
served in the biosynthesis of some other PNAs such as puromy-
4), and Mhr20 could catalyze ligation of L-arginine to amipurimy-
cinine (Figure 5); however, no cross-reactivity was observed with
respect to their amino acid substrates. These observations are con-
sistent with previous reports that ATP-grasp ligases are highly spe-
cific with respect to their amino acid substrates while being more
3
3
cin. Additional work is required to gain further insight into the
features of the biosyntheses of amipurimycin and the miharamy-
cins.
34,35
flexible regarding their acceptor substrates.
Characterization of Mhr20 and Amc18 as ATP-grasp ligases in-
volved in the biosynthesis of the miharamycins and amipurimycin. By
analogy to other known PNA biosynthetic pathways, the ATP-
grasp ligases encoded by amc18 and mhr20 were identified as likely
candidates for catalyzing amide bond formation in the biosynthesis
of amipurimycin and the miharamycins, respectively. Therefore,
their corresponding ORFs were cloned and overexpressed in E. coli
(‒)-cispentacin(16)
L-arginine
ATP
Mhr20
O
O
O
H2N
ATP
Amc18
O
O
OH
NH2 HN
OH
H
2
N
OH
O
N
O
N
HN
HO
N
HO
amc18 deletion HO
N
NH
NH
O
N
H
2
N
HO
HO
N
OH
OH
OH
OH
HO
N
N
N
N
OH
N
N
NH
2
NH2
OH
NH
2
amipurimycin (3)
Exact Mass: 495.208
amipurimycinine (19)
Exact Mass: 384.139
20
Exact Mass: 540.240
3
Amc18
3
20
m/z 496.214 [M+H]
+
m/z 541.249 [M+H]
+
as His
6
-tagged constructs prior to purification by Ni(II) affinity
(‒)-cispentacin
chromatography. In order to prepare a substrate to assay the activi-
ty of Mhr20, commercially-available peptidases were screened for
their ability to remove the pendant amino acids from the miha-
ramycins. This led to the discovery of a protease from Streptomyces
griseus that cleaves the L-arginine moiety of miharamycin B (2) to
release the core nucleoside miharamycinine (17), a putative sub-
strate for Mhr20. Likewise, single crossover mediated deletion of
amc18 in S. sp. SN-C1 provided the mutant S. sp. SN-C1Δamc18
Amc18
-arginine
19
L
Mhr20
(‒)-cispentacin
Mhr20
L
-arginine
20
1
3
5
7
9
492 494 496 498 500 502
537 539 541 543 545 547
Retention time [min]
m/z
m/z
Figure 5. Overview of in vitro assays performed using amipurimy-
cinine (19) as the amino acid acceptor; extracted ion current chroma-
4
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Page 5 of 9
Journal of the American Chemical Society
tograms of in vitro assay substrate (black trace for 19) and products
To determine the function of Mhr24, its encoding gene was re-
moved from the S. miharaensis genome through double-crossover
mediated in-frame deletion. The resulting strain, S. mi-
haraensisΔmhr24, was found to produce only miharamycin B (2).
To ensure this observation was not due to polar effects resulting
1
2
3
4
5
6
7
8
9
(red and green traces for 3 and 20, respectively) as well as mass spec-
tra of the products of the assays are shown.
5
Mhr24 is a hydroxylase involved in the biosynthesis of N -
5
hydroxyarginine. While the hydroxylated guanidine of the N -
41
from the deletion, mhr24 was cloned into vector pIB139 down-
hydroxyarginine found in the structure of miharamycin A (1) re-
sembles a key intermediate in the reaction of nitric oxide synthase,
no homolog of nitric oxide synthase could be identified in the mhr
42
stream from an ermE promoter sequence to yield the complemen-
tation vector pIB139mhr24. When S. miharaensisΔmhr24 was
transformed with pIB139mhr24, albeit not to the extent of the
wild-type strain, production of miharamycin A (1) was restored
5
cluster. Recently, N -hydroxyarginine has also been found as a
component of argolaphos A (21), a phosphonate natural product
(Figure 6D). The relatively low sequence homology between
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
36
produced by Streptomyces monomycini NRRL B-24309, and the
Mhr24 and its homolog from the argolaphos gene cluster may re-
flect the structural dissimilarity between argolaphos (21) and mi-
haramycin A (1). Considering, then, that hydroxylation of these
compounds’ respective pendant arginine moieties likely occurs late
in their biosynthetic pathways, we predict miharamycin B (2) is
generated first and is the substrate for Mhr24-mediated oxidation
to yield miharamycin A (1).
mhr cluster contains Mhr24, whose product shows homology
(24.7% similarity, 17.7% identity) to a predicted "YqcI/YcgG”
protein (NCBI accession WP_078624154) encoded in the argo-
5
laphos biosynthetic gene cluster. Although N -hydroxyarginine and
similarly hydroxylated guanidines have precedent in natural prod-
1
4,37-39
ω
ucts,
only DcsA, a YqcI/YcgG homolog and putative N -
hydroxy-L-arginine hydroxylase from Streptomyces lavendulae, has
Cispentacin biosynthesis mirrors biosynthesis of coronafacic acid.
The introduction of (–)-cispentacin (16) to the saccharide core is a
key tailoring step in amipurimycin biosynthesis. The construction
of (–)-cispentacin is presently obscure despite its importance as a
lead compound in the development of icofungipen, a potent anti-
fungal compound that inhibits eukaryotic leucyl-tRNA synthe-
been implicated in the oxidation of arginine in natural product
4
0
biosynthesis (Figure 6A). Thus, Mhr24 emerged as a reasonable
candidate for the requisite N-hydroxylase activity in the biosynthe-
5
sis of N -hydroxyarginine.
NH2
NH2
H
43-45
H
N
DcsA HN
HN
N
OH
A.
B.
C.
cycloserine
tase. While the cyclopentyl carbocycle of (–)-cispentacin is not
HN
OH
O
well-represented in nature, it bears a striking similarity to 2-
NH2
O
OH
N
ω
-hydroxy-L-arginine
carboxy-2-cyclopentenone (25, CPC), an intermediate in the bio-
L-arginine
4
6
synthesis of coronafacic acid.
OH
NH
2
NH
2
H
N
H
N
Mhr24 HN
H
N
HN
N
In the amc cluster, flanking the core set of genes shared with the
mhr cluster are amcA–H, whose products include the complete
complement of enzymes comprising the conserved biosynthetic
pathway of CPC (cfa cluster). Moreover, the amcA–H subcluster is
augmented by the presence of an enoyl reductase (AmcA) and an
acetylornithine-like aminotransferase (AmcC) that are not typically
found in CPC gene clusters. It is therefore hypothesized that the
biosynthesis of (–)-cispentacin (16) parallels the coronafacic acid
biosynthetic pathway and commences with the formation of ACP-
linked CPC (25) mediated by AmcH, B, F, G, and E, as previously
reported for the analogous sequence catalyzed by Cfa5, 1, 3, 4, and
NH
2
O
NH2
O
miharamycin B (
2
)
miharamycin A (1)
HN
NH
2
N
OH
O
P
O
H
N
NH
Me
2
HO
N
H
HO
O
Me
Miharamycin A (1)
argolaphos A (21
)
10.0
8
[
M8H] : 553.2
9.0
.0
ꢀ
D.
7.0
6.0
2
5.0
4.0
3
.0
.0
Miharamycin,B
S.#miharaensis#
2
1.0
Δ
mhr24#+, 0.01
46,47
2
00
700
300
500
2, respectively, and is concluded by the action of AmcA, AmcC,
miharamycin,B,co8injection
S.#miharaensis# mhr24#+,
pIB139mhr24
m/z
2
Δ
and the thioesterase AmcD (see Figure 7).
1
1
Miharamycin B (2)
8
7.0
[M8H] : 537.2
O
2
2
S.#miharaensis# mhr24
Δ
6.0
U-13
[
C
6
] glucose (
4)
5
.0
.0
CoAS
4
acetyl-CoA (24
)
3.0
O
CO
2
OH OH
HO
O
HO
O
S.#miharaensis#
OH
O
2
.0
.0
OH
OH
O
O
OH
O
1
O
HO
HO
7
12
Retention,time,(min)
17
0.0
O
HO
OH
OH
O
O
1
700
00
300
500
m/z
O
HO
O
O
pyruvate (22
)
oxaloacetate (23)
citrate
cis-aconitate
isocitrate
E.
Cfa5
AmcH
O
O
O
O
O
SCoA
OH
O
HO
OH
H
H
HO
SCoA
O
O
O
malonyl-CoA
CoASH
CO2
Cfa1,3
AmcB,F
succinate
semialdehyde
α-ketoglutarate (6)
AmcB
Figure 6. (A) DcsA is the proposed hydroxylase involved in arginine
AmcB
O
S
AmcB
Cfa2
AmcB
AmcB
AmcD H2
N
O
OH
ω
N -oxidation in cycloserine biosynthesis; (B) proposed function of
Cfa4
AmcG
O
S
O
O
S
AmcE
AmcA
O
S
AmcC
H2
O
S
5
Mhr24 in the oxidation of arginine N of miharamycin B (2) to yield
H
O
5
O
O
N
miharamycin A (1); (C) argolaphos
A
(21) contains N -
O
OH
(
−)-cispentacin (16)
hydroxyarginine; (D) HPLC chromatograms (λ= 305 nm) of in vivo
assays of Mhr24 demonstrate its role in the biosynthesis of miharamy-
cin A with each peak verified by MS analysis; (E) the putative argo-
laphos gene cluster encodes a YqcI/YcgG protein.
CPC (25
)
Figure 7. Proposed CPC-based (–)-cispentacin biosynthetic
13
pathway. Colored carbon atoms reflect the observed C-labeling
5
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Journal of the American Chemical Society
Page 6 of 9
pattern. The corresponding enzymes from the coronafacic acid
pathway (cfa) are shown in red.
Present Addresses
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1University of Texas M.D. Anderson Cancer Center, Investigational
Pharmacy Services, Houston, TX 77030, USA.
2
1
3
Department of Biological Engineering, Massachusetts Institute of
In support of this pathway, C NMR analysis of amipurimycin
Technology, Cambridge, MA 02139, USA
1
3
13
labeled with [U- C
6
]glucose (4) showed efficient C incorpora-
3
Department of Biochemistry and Molecular Biology, Johns Hopkins
tion into the (–)-cispentacin moiety, with C1"–C2", C3"–C4" and
C5"–C6" coupling observed. Interestingly, CPC biosynthesis be-
gins with a-ketoglutarate (6 in Figure 7), yet the labeling pattern
seen in (–)-cispentacin does not match the portion of the amipu-
rimycin core saccharide in which the a-ketoglutarate precursor (6
in Figure 3) is predicted to yield a "4+1" labeling pattern (i.e., C3'-
C4'-C5'-C6', and C7'). However, a-ketoglutarate can also be bio-
synthesized from anaplerotically generated oxaloacetate (23) using
University Bloomberg School of Public Health, Baltimore, MD 21205,
USA
4
Graduate School of Engineering, Hokkaido University, Sapporo,
Hokkaido 060-8628, Japan
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Author Contributions
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
1
3
ꢀ
pyruvate (22) derived from [U- C
6
]glucose. When this labeled
‖These authors contributed equally.
oxaloacetate is combined with acetate (24) derived from the fed
1
3
[U- C
6
]glucose, the Krebs cycle could generate "2+2" labeled a-
Funding Sources
ketoglutarate (6), leading to the pattern observed in (–)-
cispentacin (see Figure 7). Thus, it is plausible that the a-
ketoglutarate used in the core saccharide and (–)-cispentacin are
generated in different phases of growth of the producing bacteria.
Therefore, the observed (–)-cispentacin labeling pattern in this
work is consistent with previous feeding studies of the biosynthesis
This work is supported in part by grants from the National Institutes of
Health (GM035906 and GM040541) and the Welch Foundation (F-
1511) to H.-w.L.; JSPS KAKENHI (16H06453) and the Japan Agency
for Medical Research and Development (AMED) to T.K.
ACKNOWLEDGMENT
4
6,48
of coronafacic acid.
We thank Masato Tani and Makoto Ohyama of Meiji Seika Pharma
Co., Ltd., for their kind gift of authentic samples of miharamycins and
Dr. Mark Ruszczycky for his critical review of the manuscript.
Conclusion
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differ significantly from those of other, previously described PNAs
and implicate a polyketide origin for the core saccharide. Indeed,
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Corresponding Author
*To whom correspondence may be addressed. E-mail:
h.w.liu@mail.utexas.edu and utkuz@mail.ecc.u-tokyo.ac.jp
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artwork image:
Table
Of
Contents
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Amc8
Amc8
α-ketoglutarate
HO 7'
6'
KS AT ACP
KS AT ACP
O
O
O
O
Amc7
S
S
SH
S
HN
4'
HO
OH
N
O
O
O
O
O
NH
2
O
O
HO 3'
N
OH
OH
8'
HO 9'
OH
1'
1
,3-bisphospho-
glycerate
HO
N
N
ACP
S
HO
O
O
NH
2
OPO3=
amipurimycin
O
Amc9-12
O
OH
O
OH
OH
O
HO
OH
OPO3=
O
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