Discovery of (Dihydro)pyrazine N-Oxides via Genome Mining in Pseudomonas

Article abstract of DOI:10.1021/acs.orglett.8b01944

Overexpression of the Pseudomonas virulence factor (pvf) biosynthetic operon led to the identification of a family of pyrazine N-oxides (PNOs), including a novel dihydropyrazine N,N′-dioxide (dPNO) metabolite. The nonribosomal peptide synthetase responsib

Full text of DOI:10.1021/acs.orglett.8b01944

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Discovery of (Dihydro)pyrazine N‑Oxides via Genome Mining in
Pseudomonas
‡
§
Ashley M. Kretsch,† Gina L. Morgan,† Jillian Tyrrell,†,⊥ Emily Mevers, Isabelle Vallet-Gely,
́
and Bo Li*,†
†Department of Chemistry, The University of North Carolina at Chapel Hill, 250 Bell Tower Road, Chapel Hill, North Carolina
27599, United States
‡Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston,
Massachusetts 02115, United States
§
́
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Universite Paris-Saclay, 91198, Gif-sur-Yvette
cedex, France
S
* Supporting Information
ABSTRACT: Overexpression of the Pseudomonas virulence factor (pvf) biosynthetic operon
led to the identification of a family of pyrazine N-oxides (PNOs), including a novel
dihydropyrazine N,N′-dioxide (dPNO) metabolite. The nonribosomal peptide synthetase
responsible for production of (d)PNOs was characterized, and a biosynthetic pathway for
(d)PNOs was proposed. This work highlights the unique chemistry catalyzed by pvf-encoded
enzymes and sets the stage for bioactivity studies of the metabolites produced by the
virulence pathway.
Pseudomonas bacteria thrive in diverse environments and
interact extensively with eukaryotic hosts and other microbes.
These bacteria produce a variety of small molecules that
possess unique structures and functions, including side-
rophores, phytotoxins, antibiotics, and quorum-sensing mole-
cules.1−4 The genomes of these bacteria contain many
uncharacterized biosynthetic gene clusters, including the
Pseudomonas virulence factor (pvf) cluster. The pvf cluster was
identified through genetic screens and is implicated in the
virulence of the animal pathogen Pseudomonas entomophila L48
and the plant pathogen Pseudomonas syringae pv. syringae
UMAF0158.5−8 Disruption of pvfB, C, and D in P. entomophila
and P. syringae significantly decreases the virulence of these
strains against Drosophila and tomato plants, respectively.5,7
The pvf cluster was also found to be important for the
biocontrol activity of a Pseudomonas fluorescens strain.9
Additional studies found that the pvf cluster does not encode
enzymes that synthesize toxins to directly harm the host, but
rather synthesize signaling molecule(s) that regulate virulence
factors, including the pore-forming toxin monalysin in P.
entomophila and the phytotoxin mangotoxin in P. syringae.5,8,10
The pvf operon contains four genes: a putative nonribosomal
peptide synthetase (NRPS, pvf C), a putative diiron N-
oxygenase (pvfB), and two genes of unknown function (pvfA
and pvfD, Figure 1A, Table S1). NRPSs are a large family of
multidomain enzymes that are responsible for the production
of many bioactive natural products such as the antibiotics
vancomycin and daptomycin and the virulence factor
pyoverdine.11−13 PvfB shares homology with AurF and CmlI,
which convert aryl amines into aryl nitro compounds in the
biosynthesis of the antibiotics aureothin and chloramphenicol,
respectively.14−17
Homologues of pvfA−D were identified in Burkholderia
cenocepacia H111 as part of the ham cluster (hamA−G, Figure
S1), which was recently shown to be responsible for the
synthesis of fragin, an antifungal molecule.18 Deletion of the
fragin biosynthetic gene hamF that is absent in the pvf cluster
led to the discovery of valdiazen, a valinol diazeniumdiolate.
Valdiazen exhibits moderate signaling activity toward the
hamA promoter reporter strain and was proposed to be the
signaling molecule that autoregulates the expression of ham
genes.18 However, this study also found that valdiazen could
not be identified in pvf-containing Pseudomonas strains that do
not contain hamF or hamG homologues.18 Therefore, we
propose pvf-encoded enzymes from Pseudomonas produce
different bioactive molecule(s) than valdiazen and set out to
identify these small molecules using P. entomophila as a model.
Through overexpression of the pvf operon, we identified and
characterized a new family of natural products, (dihydro)-
pyrazine N-oxides ((d)PNOs). We found that PvfC and PvfB
are responsible for the biosynthesis of this unique family of
compounds. With this evidence, we propose a biosynthetic
Received: June 21, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b01944
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spectroscopies (Figure 2, Table 1, Tables S4−S5, Figures S3−
22).
Figure 2. Structural characterization of dPNO (1), PNO A (2), and
PNO B (3). COSY correlations are indicated by bonds in bold, and
key (1H, 13C)-HMBC and (1H, 15N)-HMBC correlations are
indicated by red and blue arrows, respectively.
Figure 1. Identification of new metabolites produced by pvf-encoded
enzymes using comparative metabolomics. (A) The pvf biosynthetic
cluster from P. entomophila. (B) Overexpression of pvfABCD on a
pPSV35 vector in P. entomophila Δpvf C (green) led to the production
of three new metabolites 1−3, which are absent from P. entomophila
Δpvf C (red). These metabolites were also identified when pvfABCD
was heterologously expressed in P. aeruginosa PAO1 (blue).
The 1H spectrum in conjunction with the molecular formula
indicates that 2 is symmetric. The COSY correlations between
six methyl protons and a single multiplet proton indicate the
presence of an isopropyl group (Figure 2). The shift at δH 8.09
and UV spectra suggest a heterocyclic pyrazine core (Table S4,
Figures S3, S13).20 The oxygens are likely attached to pyrazine
as N-oxides. Together, the structure of 2 was assigned as 2,5-
diisopropylpyrazine N,N′-dioxide (PNO A).
pathway for (d)PNOs and suggest a shared biosynthetic
intermediate for (d)PNOs and valdiazen.
Many bacterial small molecules, especially signaling
molecules, are natively produced at low levels.19 To enhance
the production of small molecules made by pvf-encoded
enzymes, we engineered an inducible expression system for the
pvf cluster and identified small molecules unique to pvf-
expressing strains. The expression plasmid containing
pvfABCD under an IPTG-inducible lacUV5 promoter was
introduced into a P. entomophila mutant from which the NRPS
gene pvfC has been deleted (ΔpvfC),5 generating the native
pvf overexpression strain (ΔpvfC + pPSV-pvfABCD). The
culture supernatants of this pvf expression strain and the
ΔpvfC control were extracted using dichloromethane. The
extracts were analyzed using liquid chromatography-coupled
high-resolution mass spectrometry (LC-HRMS). Comparison
of the metabolic profiles revealed several unique compounds.
Three major metabolites are present at high levels in the
extract of ΔpvfC + pPSV-pvfABCD, but are absent in the
extract of the ΔpvfC control: 1 ([M + H]+, 199.145 m/z,
C10H18N2O2), 2 ([M + H]+, 197.126 m/z, C10H16N2O2), and
3 ([M + H]+, 181.135 m/z, C10H16N2O) (Figures 1B, S2). The
pvf plasmid was also introduced into Pseudomonas aeruginosa
PAO1, which does not natively harbor the pvf cluster, yielding
a heterologous pvf expression strain (PAO1 + pPSV-
pvfABCD). Metabolites 1−3 were also detected in the organic
extract of PAO1 + pPSV-pvfABCD, but not in the extract of the
control strain carrying the empty expression vector (Figures
1B, S2). These results indicate that 1, 2, and 3 are synthesized
by the pvf-encoded enzymes.
The chemical formula of 3 contains one less oxygen than 2,
and the NMR spectrum suggests that 3 is asymmetric. The 1H
shifts of 3 are largely similar to those of 2, suggesting 3
possesses a related structure but contains a single N-oxide. The
COSY, (1H, 13C)-HMBC, and (1H, 15N)-HMBC NMR
correlations support the pyrazine core and were used to assign
the isopropyl groups to the 2,5 positions of the pyrazine
(Figure 2, Table S5). The structure of 3 was assigned as 2,5-
1
diisopropylpyrazine N-oxide (PNO B). The H and 13C shifts
of PNO A and PNO B are consistent with the shifts reported
for these compounds, which have been previously synthe-
sized.21−24
The chemical formula indicates that 1 has one less degree of
unsaturation than 2 and 3, which may be due to hydration
within the pyrazine ring. The 1H NMR spectrum of 1 contains
an additional multiplet proton at δ3.89 and two diastereotopic
protons at δ4.16 and δ4.49 (Table 1). The connectivity of
these atoms was determined based on COSY, (1H, 13C)-
HSQC, (1H, 13C)-HMBC, and (1H, 1H)-TOSCY NMR
correlations. These correlations and proton splitting patterns
support the presence of a 2,3-dihydropyrazine core, and the
(1H,15N)-HMBC correlations indicate the positions of 2,5-
diisopropyl groups (Table 1, Figure 2). Thus, the structure of 1
was assigned as 2,5-diisopropyl-2,3-dihydropyrazine N,N′-
dioxide (dPNO).
We isolated 1, 2, and 3 from 8 L cultures of ΔpvfC + pPSV-
pvfABCD for structure elucidation. The major metabolites
were isolated at a yield of 5 mg/L for 1 and 1−2 mg/L for 2
and 3. The structures of these metabolites were analyzed and
characterized using a combination of NMR, UV, and IR
The structure of 1 contains two conjugated nitrones and is
highly unusual. We conducted UV absorbance and IR
spectroscopy to further characterize this molecule. We found
that 1 exhibits two UV absorbance maxima at 219 and 348 nm
(Figure S3) and distinctive IR peaks at 1564, 1435, and 1398
B
DOI: 10.1021/acs.orglett.8b01944
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Table 1. NMR Assignments and Correlations for dPNO (1)
position
δC
proton
δH (multiplicity, J [Hz])
COSY
(1H, 13C) HMBC
δN
(1H, 15N) HMBC
1-N
2
3a, b
4-N
5
6
7
8, 9
10
11
12
−94 (286)
72.0
57.7
CH
CH2
3.89 (td, [6.5, 4.4])
4.49 (dd, [15.9, 6.8]), 4.16 (dd, [15.9, 4.4])
3a, 3b
2, 3b, 3a
6, 10, 11, 12
2, 5, 6, 10
1, 4
1, 4
−121 (259)
145.2
126.8
26.4
18.4, 18.7
28.9
CH
CH(CH3)2
CH3
CH(CH3)2
CH3
CH3
7.39 (s)
2, 5, 7
1, 4
4
3.42 (hept, [7.0])
1.15 (d, [7.0])
2.48 (m, [13.5, 6.8])
1.09 (d, [7.1])
1.03 (d, [6.8])
8, 9
7
11, 12
10
5, 6, 8, 9
5, 7, 8, 9
2, 3, 11, 12
2, 10, 12
2, 11, 10
1
19.0
17.7
10
cm−1 (Figure S5), which is consistent with the reported UV
maxima in the 345−370 nm range and strong IR bands at
1530−1570 cm−1 and 1440−1470 cm−1 for synthetic dinitrone
compounds.25−27 These results further support the proposed
structure for 1. The stereochemistry at C2 remains to be
determined.
We synthesized 2 and 3 to confirm the proposed structures
and obtain materials for biological studies (Scheme S1).
Products 2 and 3 were synthesized starting with L-valinol to
yield a 2,5-diisopropylpyrazine intermediate that was oxidized
1
to yield 2 and 3. The H and 13C NMR and UV spectra of
synthetic 2 and 3 are consistent with previous reports21−23 and
are nearly identical to the spectra for 2 and 3 isolated from P.
entomophila (Figures S23−27, Tables S4−S5), confirming the
structural assignments.
We conducted biochemical and genetic studies to character-
ize the biosynthesis of (d)PNOs. Overexpression of different
combinations of pvf genes revealed that pvfB and pvfC are
necessary and sufficient for the production of (d)PNOs
(Figure S28). The NRPS PvfC contains three domains:
adenylation (A), thiolation (T), and reductase (R) (Table S1,
Figure 3A). The adenylation activity of PvfC was investigated
in vitro using a ATP-[32P]PPi exchange assay. PvfC selectively
activates L-Val among the 20 proteinogenic amino acids (KM =
3.4 0.7 mM, kcat = 3.4 0.3 s−1, Figures 3A and B, S29).
The observed KM is 2−3-fold higher than that of typical NRPS
adenylation domains,28 but is generally consistent with the mM
cellular concentration of L-Val in bacteria.29 The incorporation
of Val was also investigated in cells using isotopically labeled
Val ([2H1−8]DL-Val). An increase of +7 and +14 (and +15 for
1) was observed for 1, 2, and 3 (Figures 3C, S30), when the
overexpression cultures were supplemented with [2H1−8]DL-
Val, indicating the incorporation of one or two [2H1−8]DL-Val.
This result suggests that L-Val is the physiological substrate for
PvfC and is likely the precursor for (d)PNOs.
A PvfB homologue from P. syringae pv. phaseolicola was
previously reported to convert p-aminobenzoic acid to p-
nitrobenzoic acid.30 However, neither (d)PNOs nor valdiazen
contains a p-nitrobenzoic acid. Thus, p-aminobenzoic acid may
not be the physiological substrate for PvfB. The structures of
(d)PNOs and valdiazen suggest that a N-hydroxy-L-Val
derivative exists as a precursor to both sets of molecules.
PvfB may install the hydroxylamine on PvfC-linked L-Val
instead.
Figure 3. PvfC activates and incorporates L-Val into dPNO (1), PNO
A (2), and PNO B (3). (A) The adenylation domain of PvfC activates
and loads L-Val onto the thiolation domain. (B) Kinetic measure-
ments of PvfC with L-Val. KM = 3.4 0.7 mM, kcat = 3.4 0.3 s−1.
(C) Incorporation of [2H1−8]DL-Val into (d)PNOs based on LC/
HRMS analysis. Domain abbreviations: adenylation (A), thiolation
(T), and reductase (R).
hydroxy-L-Val linked to the T domain by PvfC R domain
releases the intermediate as an aldehyde. Two molecules of this
aldehyde cyclize and dehydrate to create the 2,4-dihydropyr-
azine N,N′-dioxide. Isomerization forms dPNO, while
aromatization forms PNO A. Production of the monoxide
PNO B may result from transformation from dPNO (Figure
S31) or cyclization between a N-hydroxy-L-Val aldehyde and a
L-Val aldehyde. Valdiazen produced by Burkholderia may result
from a four-electron reduction of the T-domain-linked N-
hydroxy-L-Val to generate N-hydroxy-L-valinol. This molecule
can be further modified to the diazoniumdiolate (Figure 4).
We evaluated the activity of (d)PNOs to restore the
virulence of the Δpvf C mutant using a fly infection assay.5
Cultures of the ΔpvfC mutant were supplemented with
isolated 1, isolated or synthetic 2, or isolated or synthetic 3.
After 24 h of growth, these cultures were concentrated and fed
to adult female flies. These molecules do not restore the
We propose a biosynthetic pathway for (d)PNOs (Figure
4). First, PvfC activates and loads L-Val onto the T domain.
The amine of the tethered L-Val is oxygenated by PvfB to
generate a hydroxylamine. A two-electron reduction of the N-
C
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Figure 4. Proposed biosynthesis of (dihydro)pyrazine N-oxides in P. entomophila L48 and valdiazen in B. cenocepacia H111.
virulence of ΔpvfC against adult flies individually or combined
(Figure S32), while the extracts of the P. entomophila wildtype
culture restored the virulence of ΔpvfC to wild type levels.5
Furthermore, the biosynthesis of (d)PNOs does not require
PvfD (Figure S28). Previously, pvf D and a homologue were
shown to be essential for signaling activity.5,7 Together, the
evidence suggests that (d)PNOs are potential biosynthetic
intermediates or shunt products of the pvf pathway. As some
signaling pathways require the synergistic action of multiple
metabolites,31,32 additional metabolites produced by pvf-
encoded enzymes may be necessary for the full signaling
activity. Identifying the additional metabolites required for the
signaling activity will be the focus of future studies.
Although (d)PNOs do not restore the signaling activity of
Δpvf C, their unique structures suggest other potential
activities toward microbes or multicellular organisms. Volatile
pyrazines are well-established pheromones for insects and are
distributed in environmental niches ranging from Pseudomonas
spp. on wine corks to bacteria associated with leaf cutter
ants.33−38 Recently, a 3,5-dimethylpyrazin-2-ol was identified
as a quorum-sensing molecule for Vibrio cholerae.39 In regard
to the N-oxides, only a few examples have been identified in
natural products, such as 4-hydroxy-2-heptylquinoline-N-oxide
(HQNO) and iodinin (1,6-dihydroxy phenazine N,N'-
dioxide), which possess signaling, antimicrobial, and redox
cycling activities.40−43 The N-oxide derivatives of pyrazines are
rare; in particular, the 2,3-dihydropyrazine N,N′-dioxide
moiety of 1 is unprecedented in natural products, to the best
of our knowledge. Unlike iodinin, dPNO does not have
connected aromatic rings, yet it is stable at the pH range 1−11
(Figure S33) and for several months at room temperature (less
than 15% was converted to PNO B after one year in storage,
Figure S31). Attempts to synthesize dPNO have been
unsuccessful; however, overexpression of pvf allows the
isolation of this unique compound in relatively high yields
for further exploration of its chemical properties and biological
activities.
biosynthesis of a dityrosyl-pyrazine in Aspergillus flavus.45
Interestingly, a N,N′-dioxide species of dityrosyl-pyrazine has
also been isolated, but no PvfB homologue was found in the
gene cluster and the biosynthetic origin of this N-oxidation is
unknown. Reactions conducted by PvfC and PvfB and their
homologues provide essential chemistries for divergent biosyn-
thesis of a variety of structures, including diazeniumdiolates in
fragin and valdiazen and (dihydro)pyrazine N-oxides.
In summary, through genome-mining in Pseudomonas and
overexpression of the pvf gene cluster, we have identified and
characterized a family of pyrazine N-oxides, including a novel
dihydropyrazine N,N′-dioxide metabolite. This work identifies
the role of a noncanonical NRPS in the biosynthesis of
pyrazine derivatives and suggests a common biosynthetic
origin for two families of molecules. Our work sets the stage for
understanding the biosynthesis and bioactivity of (d)PNOs
and identifying new molecules involved in Pseudomonas
signaling and virulence.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b01944.
Detailed experimental procedures, supplemental figures,
and spectral data (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: boli@email.unc.edu.
ORCID
Bo Li: 0000-0002-8019-8891
Present Address
⊥Truvian Sciences, 4535 Towne Centre Court, Ste 201, San
Diego, CA 92121, United States.
We have identified a new biosynthetic pathway for pyrazine
derivatives involving a noncanonical NRPS, PvfC, and an iron-
dependent N-oxygenase, PvfB. The A-T-R domain structure of
PvfC resembles two fungal NRPSs, one involved in a general
biosynthetic pathway for lysine44 and the other specific for the
Notes
The authors declare no competing financial interest.
D
DOI: 10.1021/acs.orglett.8b01944
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ACKNOWLEDGMENTS
■
The authors thank Albert Bowers (UNC Chapel Hill) for
assistance with synthesis and Jon Clardy (Harvard Medical
School) for helpful discussions and Alex Miller and Quinton
Bruch (UNC Chapel Hill) for assistance with IR. This work is
supported by the National Institutes of Health
(R00GM099904 and DP2HD094657 to B.L.), the Rita Allen
Foundation, and the University of North Carolina at Chapel
Hill. E.M. acknowledges support from NIH Grant
R01AT009708 to J.C. A.M.K. acknowledges support from a
Burroughs Wellcome Graduate Fellowship.
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