Argicyclamides A-C Unveil Enzymatic Basis for Guanidine Bis-prenylation

Article abstract of DOI:10.1021/jacs.1c05732

Guanidine prenylation is an outstanding modification in alkaloid and peptide biosynthesis, but its enzymatic basis has remained elusive. We report the isolation of argicyclamides, a new class of cyanobactins with unique mono- and bis-prenylations on guanidine moieties, from Microcystis aeruginosa NIES-88. The genetic basis of argicyclamide biosynthesis was established by the heterologous expression and in vitro characterization of biosynthetic enzymes including AgcF, a new guanidine prenyltransferase. This study provides important insight into the biosynthesis of prenylated guanidines and offers a new toolkit for peptide modification.

Full text of DOI:10.1021/jacs.1c05732

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Argicyclamides A−C Unveil Enzymatic Basis for Guanidine Bis-
prenylation
Chin-Soon Phan,# Kenichi Matsuda,# Nandani Balloo, Kei Fujita, Toshiyuki Wakimoto,*
and Tatsufumi Okino*
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ABSTRACT: Guanidine prenylation is an outstanding modification in alkaloid and peptide biosynthesis, but its enzymatic basis has
remained elusive. We report the isolation of argicyclamides, a new class of cyanobactins with unique mono- and bis-prenylations on
guanidine moieties, from Microcystis aeruginosa NIES-88. The genetic basis of argicyclamide biosynthesis was established by the
heterologous expression and in vitro characterization of biosynthetic enzymes including AgcF, a new guanidine prenyltransferase.
This study provides important insight into the biosynthesis of prenylated guanidines and offers a new toolkit for peptide
modification.
yanobactins are a class of ribosomally synthesized and
post-translationally modified peptides (RiPPs) produced
micropeptins and kawaguchipeptins (4, 5) (Figure 1).20−22
The LCMS profile of compounds 1−3 showed molecular ion
peaks at m/z 1058, 990, and 922, respectively (Figure S1).
This is reminiscent of a homologous series that differs by 68
atomic mass units, and this difference corresponds to a prenyl
group. The HRESI(+)MS analysis of purified 1 yielded a
molecular ion [M + H]+ at m/z 1058.7128, indicative of the
C
by cyanobacteria.1−3 Although their chemical structures are
hypervariable in terms of amino acid sequences, cyanobactins
are biosynthesized through a common pathway.4,5 Cyanobac-
tins are directly encoded in the C-terminal region of the
precursor peptide (IPR031036) as the core peptide. The core
peptide is flanked by recognition sequences (RSs) that
facilitate proper recognition from biosynthetic enzymes
modifying the core peptide. The universal proteolytic step
that removes the region preceding the core peptide (leader
peptide) is mediated by subtilisin-like S8A protease
(IPR023830) represented by PatA.6 Subsequently, a second
subtilisin-like S8A protease represented by PatG removes the
region following the core peptide (follower peptide) to yield
head-to-tail cyclic or linear cyanobactins.6,7 In many cases, the
core peptide undergoes divergent modification steps including
heterocyclization to generate oxazol(in)e/thiazol(in)e,8−11
methylesterification on C-terminal carboxylic acid,7 and
forward or reverse prenylation and geranylation.12−17 The
prenylation is catalyzed by ABBA-type prenyltransferases
(PTases, IPR031037) that are selective for the isoprenyl
donor (dimethylallyl diphosphate: DMAPP or geranyl
diphosphate: GPP) and the residue that is prenylated.12−17
On the other hand, they exhibit extremely relaxed specificity
against the remainder of the prenyl acceptor, and thus
cyanobactin PTases could be versatile biochemical tools for
late-stage peptide modifications.12,18 The catalytic variations of
cyanobactin PTases revealed that the residues prenylated by
these PTases are not only Tyr, Ser, Thr, and Trp12−17 but also
N- and C-termini of linear cyanobactins.7,19 However, the
prenylation sites are limited to these residues, and the
prenylation of a charged side chain has not been observed.
During the course of our efforts toward the discovery of
natural products, by targeting cyanobacteria from the NIES
collection, we found the unreported compounds 1−3 from the
well-studied strain M. aeruginosa NIES-88, a producer of
molecular formula C57H91N11O8 (Δ0.3 mDa). The 1H and 13
C
NMR spectra of 1 in CD3OD (Table S1) showed eight signals
for the Cα methines of α-amino acids (δC 66.5, 63.5, 62.4,
61.1, 59.8, 58.4, 57.2, 51.7; δH 4.66, 4.55, 4.24, 4.23, 4.23, 4.09,
3.78, 2.99), and six signals for amide NH protons (Figure S37),
probably due to slow exchange to deuterium through inter-
residual hydrogen bonding. In addition, signals for an N-prenyl
unit were also observed, and two sets of them are overlapped
according to the integral intensity (δC 138.6, 119.8, 40.7, 25.8,
18.1; δH 5.22, 3.82, 1.76, 1.71). A detailed analysis of the 2D
NMR data established that 1 is the octapeptide composed of
Phe, Ile, Val (× 2), Leu, Pro (× 2), and Arg residues (Figure
2). The sequence of these residues was determined by
interpretations of the HMBC and ROESY spectra in
CD3OD, which allowed the assignment of two partial
structures: the dipeptide Arg-Val(1) and the hexapeptide
Phe-Ile-Val(2)-Leu-Pro(1)-Pro(2) (Figure 2a). It was difficult
to connect these two fragments, due to the overlapped signals
for the α-methine protons of Arg and Val(1) at δH 4.23 in
CD3OD (Figure 2a). These two fragments were connected
based on the ROESY correlations in DMSO-d6 (Figure 2b),
which allowed us to close the linear fragments into a head-to-
Received: June 4, 2021
Published: June 28, 2021
https://doi.org/10.1021/jacs.1c05732
J. Am. Chem. Soc. 2021, 143, 10083−10087
© 2021 American Chemical Society
10083

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Figure 1. Structures of cyanobactins from M. aeruginosa NIES-88. Argicyclamides A−C (1−3) are newly reported in this study.
Marfey’s analysis of 3 confirmed that the macrocyclic scaffold
exclusively consists of L-amino acids (Figure S3). The structure
of the cyclic scaffold was further confirmed by total synthesis of
3, using conventional solid phase peptide synthesis, followed
by ring closure with a coupling reagent (Figure S4).24−26 While
1−3 were not cytotoxic, 1 inhibited growth of Staphylococcus
aureus, methicillin-resistant S. aureus and Bacillus subtilis with
MIC of 3.12−6.25 μM. Notably, as the number of prenyl
groups increases from one to two, the antimicrobial activity
was significantly enhanced (Table S3, Figures S5−S10).
The unique N-prenylation of 1 prompted us to investigate
its biosynthetic mechanism. As the cyclic scaffolds of
argicyclamides exclusively consist of L-amino acids, we
hypothesized that these are ribosomally synthesized peptides.
Based on this assumption, we searched for a putative precursor
peptide of argicyclamides in the genome of M. aeruginosa
NIES-88 by tBLASTn with their possible peptide backbone
sequence (RVFIVLPP) as a query; however, the corresponding
gene was not found in the public genome data. Therefore, we
resequenced M. aeruginosa NIES-88 by using both long and
short read sequencers. De novo hybrid assembly generated a
complete sequence of a 5.5 Mb chromosome and two
additional small plasmids. Scanning the whole genome
sequence identified two copies of putative precursor peptide
genes of argicyclamide, agcE1 and agcE2, which are encoded in
the remote genetic loci (Figure 3a, Figures S11 and S12).
While no cyanobactin-related genes were encoded in the
Figure 2. Key 2D NMR correlations of 1 in CD3OD (a) and DMSO-
d6 (b).
flanking region of agcE1, a putative enzyme AgcF, which shares
homology with cyanobactin PTases such as Trp C-prenyl-
transferase KgpF (AAid, 41%),14 Trp N-prenyltransferase AcyF
(AAid, 41%),15 and Nα-prenyltransferase MusF2 (AAid,
tail cyclic octapeptide. The two prenyl units are attached to the
35%),19 was found in the flanking region of agcE2. However,
although the cyclic scaffolds of argicyclamides should be
constructed by a set of PatA/G-like proteases, no such
proteases were encoded in the vicinity of the agcE1 nor agcE2.
This led us to hypothesize that AgcEs are processed by
putative PatA/G-like proteases that are encoded in remote
genetic loci. To search them, the complete genome of M.
aeruginosa NIES-88 was analyzed with HMM-based annota-
tion.27 This validated that KgpA and KgpG, proteases in
kawaguchipeptin biosynthesis, are the only PatA/G-like
proteases (IPR023830) in M. aeruginosa NIES-88. Notably,
we detected two sets of near-identical kawaguchipeptin
biosynthetic gene clusters (kgp1 and kgp2, Figure 3a) that
reside in remote loci in M. aeruginosa NIES-88 chromosome
(Figures S13 and S14). Genes coding for B, E, F, G proteins
are identical between kgp1 and kgp2. On the other hand, while
kgpA2 encodes the full-length of PatA-like protease, the N-
terminal region of kgpA1 encoding catalytic triad is truncated,
two Nωs of guanidine in Arg, as deduced from the DQF
1H−1H COSY (in DMSO-d6) correlations between NωH (δH
7.42) and CH2 (δH 3.75), and NδH (δH 7.37) and CH2 (δH
3.11), as well as the HMBC correlation between Cω and CH2
(δH 3.75) (Figure 2b). The symmetrical pattern of bis-
prenylation on Nω agrees with the overlapped signals of two
prenyl groups, thus excluding the possibility of an Nω,Nδ-
asymmetric bis-prenylated guanidine. The amide bond
between two Pro residues was assigned to be cis conformation
based on ROESY correlations and diagnostic carbon chemical
shifts (ΔδCβ − δCγ).23
Compounds 2 and 3 have the molecular formulas of
C52H83N11O8 (Δ −0.4 mDa) with [M + H]+ at m/z 990.6495,
and C47H75N11O8 (Δ0.0 mDa) with [M + H]+ at m/z
922.5873, respectively. Detailed analysis of the 2D NMR
spectra revealed that 2 and 3 possess the same macrocyclic
scaffold as 1, except that 2 has only one prenyl unit at Arg,
while 3 lacks prenylation at Arg (Figure S2, Table S2). The
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prenyltransferase AgcF was expressed in E. coli and
characterized in vitro, using dimethylallyl diphosphate
(DMAPP) as a prenyl donor and synthetic 3 as an acceptor.
A time-course analysis revealed the initial generation of 2, the
monoprenylated product, followed by the accumulation of 1,
the bis-prenylated product (Figure 4a). This demonstrated that
Figure 3. Genetic basis of cyanobactin biosyntheses in M. aeruginosa
NIES-88. (a) Cyanobactin-related genes encoded in remote genetic
loci of M. aeruginosa NIES-88 chromosome. Genes are labeled with
locus numbers. The locus tag prefix (MAN88) was omitted.
Homologous genes are linked and amino acid identities are shown
for each linkage. (b) Primary sequences of the precursor peptides
AgcEs and KgpEs, with the core peptide (bold) and proposed
recognition sequences RSII and RSIII (underlined). (c) Heterologous
production of 3 in E. coli. Extracted ion chromatograms for m/z
922.5000 are shown.
thus KgpA1 is likely to be inactive (Figure S15). Based on
these observations and the fact that the RSII (recognition
sequence of PatA-lile protease) and RSIII (recognition
sequence of PatG-like protease) of AgcEs are similar to
those of KgpE, a kawaguchipeptin precursor peptide (Figure
3b), we hypothesized that AgcEs could be matured by KgpA2
and KgpG, proteases for kawaguchipeptin biosynthesis.
To test this hypothesis, we coexpressed the precursor
peptides agcE and kgpA2/kgpG in the heterologous host E. coli
BL21 (DE3). LC-MS analyses of the transformant’s metabo-
lites revealed the production of 3, the nonprenylated cyclic
peptide (Figure 3c). Omitting kgpA2 or kgpG gene resulted in
the loss of 3 production. To further access the production of 3
in vitro, recombinant KgpG was incubated with a synthetic
AgcE-core with a follower peptide (RVFIVLPPFAEDGAE).
This resulted in KgpG-dependent production of 3 (Figure
S19). These results demonstrated substantial promiscuity of
kawaguchipeptin proteases and suggest that these are
employed for constructing the cyclic scaffolds of both
argicyclamides and kawaguchipeptins, which are structurally
distinct.
Figure 4. In vitro characterization of AgcF and proposed biosynthetic
schemes of argicyclamides (1−3) and kawaguchipeptins (4, 5). (a)
AgcF catalyzed sequential prenylations to convert 3 to 1, with the
intermediacy of 2. Extracted ion chromatograms for 1 (m/z
1058.5000, red), 2 (m/z 990.5000, blue), and 3 (m/z 922.5000,
black) are shown. (b) KgpA2/G are shared with the agc and kgp
pathways to afford distinct cyclic scaffolds (3, 5), which are tailored
by specialized PTases.
AgcF catalyzes two rounds of prenylation on the guanidine
moiety. This conversion is Mg2+-dependent and is optimal at
37 °C, pH 8.0 (Figures S20 and S21). AgcF showed no
prenylation activity on synthetic analogs of 3 with Arg
substituted to Trp, Tyr, Ser, Thr, or Lys (6−10, Figure
S22). No prenylation was observed for linear analogs (11 and
12, Figure S22). These results show that AgcF is highly
selective for 3 as a prenyl acceptor. Next, the specificity of
AgcF on the isoprenyl donor was assessed by using geranyl
diphosphate (GPP) and farnesyl diphosphate (FPP). Contrary
to other cyanobactin PTases that are highly selective for the
isoprenyl donor,12−16,28 AgcF was capable of catalyzing the
monogeranylation of 3 (Figures S23 and S24), highlighting its
Next, we investigated the biosynthetic origin of the bis-
prenylated guanidine moiety in 1. To this end, the putative
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unusual tolerance. FPP was not accepted as an isoprenyl
donor.
AUTHOR INFORMATION
Corresponding Authors
■
To gain structural insights into AgcF catalysis, we generated
a computational model of AgcF, using the crystal structure of
PagF, an O-Tyr PTase in prenylagaramide biosynthesis, as the
template (AAid, 42%) (Figure S25).13 The Mg2+ and
diphosphate binding site, as well as the proposed catalytic
residue (Glu49 in AgcF and Glu51 in PagF) that activates the
prenyl acceptor, are well conserved between AgcF and PagF.
The substitution of Glu49 to Ala in AgcF abolished its
prenylating activity, showing the general importance of this
residue in the catalysis of cyanobactin PTases (Figure S26).
Stark differences between AgcF and PagF were observed in the
residues forming the active site entrance, where the bulky
residues in PagF are substituted with substantially smaller
residues in AgcF, for example, F69/G67, H138/G133, W271/
C267, and Y292/L289, respectively (Figure S25). The
enlarged active site should facilitate the accommodation of a
bulky substrate and enable the sequential bis-prenylation of
guanidine.
A phylogenetic analysis revealed that cyanobactin PTases
form clades according to their chemoselectivities (Figure S27).
Notably, AgcF composes a small but distinct clade together
with its close homologues, and all share the aforementioned
substitutions at the active site entrance (Figure S28). The
putative Arg-containing precursor peptides encoded in the
neighboring regions of AgcF-like PTases suggest the presence
of a new class of cyanobactins, with a bis-prenylated Arg
residue yet to be identified (Figure S29).
Toshiyuki Wakimoto − Faculty of Pharmaceutical Sciences,
Hokkaido University, Sapporo 060-0812, Japan; Global
Station for Biosurfaces and Drug Discovery, Hokkaido
University, Sapporo 060-0812, Japan; orcid.org/0000-
0003-2917-1797; Email: wakimoto@pharm.hokudai.ac.jp
Tatsufumi Okino − Graduate School of Environmental
Science and Faculty of Environmental Earth Science,
Hokkaido University, Sapporo 060-0810, Japan;
orcid.org/0000-0002-8363-0467; Email: okino@
ees.hokudai.ac.jp
Authors
Chin-Soon Phan − Faculty of Environmental Earth Science,
Hokkaido University, Sapporo 060-0810, Japan;
orcid.org/0000-0002-6500-696X
Kenichi Matsuda − Faculty of Pharmaceutical Sciences,
Hokkaido University, Sapporo 060-0812, Japan; Global
Station for Biosurfaces and Drug Discovery, Hokkaido
University, Sapporo 060-0812, Japan; orcid.org/0000-
0002-9269-688X
Nandani Balloo − Graduate School of Environmental Science,
Hokkaido University, Sapporo 060-0810, Japan
Kei Fujita − Faculty of Pharmaceutical Sciences, Hokkaido
University, Sapporo 060-0812, Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c05732
In this study, we discovered argicyclamides (1−3), a new
group of cyanobactins with a unique bis-prenylated Arg
residue. Based on the complete genome sequence and series of
biochemical analyses, we proposed a unique biosynthetic route
for 1−3, in which the precursor peptide is processed by
distantly encoded maturation proteases participating in a
distinct cyanobactin biosynthetic pathway (Figure 4b). In
general, cyanobactin maturation proteases act on single or
multiple precursor peptides encoded in neighboring genetic
loci.4,5,9,16,29,30 However, to our knowledge, cyanobactin
proteases processing distantly encoded precursor peptides
have not been previously reported.
Notably, Pancrace et al. recently reported the first
biosynthetic investigation of prenylated guanidines on
aeruginoguanidines/microguanidines, a group of cytotoxic
nonribosomal peptides produced by Microcystis.31 Although
not validated experimentally, guanidine prenylation is
proposed to be catalyzed by AgdJ that belongs to the
decaprenyl diphosphate synthase-like family (IPR001441),
which shares no sequence homology with AgcF, suggesting
that several enzyme families have evolved convergently to
achieve guanidine prenylation. AgcF, a newly identified PTase
in this study is, to our knowledge, the first guanidine PTase
with biochemical validation.
Author Contributions
#C.-S.P. and K.M. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partly supported by the Asahi Glass
Foundation, the Naito Foundation, the Uehara Memorial
Foundation, the Sumitomo Foundation−Grant for Basic
Science Research Projects, Daiichi Sankyo Foundation of
Life Science, Global Station for Biosurfaces and Drug
Discovery, a project of Global Institution for Collaborative
Research and Education at Hokkaido University, the Japan
Agency for Medical Research and Development (AMED Grant
Number JP19ae0101045), the Japan Science and Technology
Agency (JST Grant Numbers ACT-X JPMJAX201F and A-
STEP JPMJTR20US) and Grants-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT),
Japan (JSPS KAKENHI Grant Numbers JP16703511,
JP18056499, and JP19178402). C.-S.P. is a recipient of the
JSPS Postdoctoral Fellowship for Foreign Researchers (ID No.
P19096).
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c05732.
Synthetic procedure, NMR, LC-MS, HPLC analysis,
strains, oligonucleotides, plasmids, genome sequence,
bioinformatics, biological activity assay, detailed meth-
ods, and materials (PDF)
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NOTE ADDED AFTER ASAP PUBLICATION
This paper was published on June 28, 2021. Figure 4 has been
updated and the paper was re-posted on June 29, 2021.
■
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