Diketopiperazine Formation in Fungi Requires Dedicated Cyclization and Thiolation Domains

Article abstract of DOI:10.1002/anie.201909052

Cyclization of linear dipeptidyl precursors derived from nonribosomal peptide synthetases (NRPSs) into 2,5-diketopiperazines (DKPs) is a crucial step in the biosynthesis of a large number of bioactive natural products. However, the mechanism of DKP format

Full text of DOI:10.1002/anie.201909052

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Accepted Article
Title: Diketopiperazine Formation in Fungi requires Dedicated
Cyclization and Thiolation Domains
Authors: Frank C Schroeder, Joshua Baccile, Henry Le, Brandon
Pfannenstiel, Jin Woo Bok, Christian Gomez, Eileen
Brandenburger, Dirk Hoffmeister, and Nancy Keller
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201909052
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Diketopiperazine Formation in Fungi requires Dedicated
Cyclization and Thiolation Domains
Joshua A. Baccile[a]†, Henry H. Le[a], Brandon T. Pfannenstiel[b], Jin Woo Bok[b], Christian Gomez[a],
Eileen Brandenburger[c], Dirk Hoffmeister[c], Nancy P. Keller[b]*, and Frank C. Schroeder[a]*
Abstract: Cyclization of linear dipeptidyl precursors derived from
nonribosomal peptide synthetases (NRPSs) into 2,5-
vivo (blue arrows, Figure 1a), functional analysis of the core
enzyme, GliP, is incomplete (Figure 1b).[14]
diketopiperazines (DKPs) is a crucial step in the biosynthesis of a
large number of bioactive natural products. However, the
mechanism of DKP formation in fungi has remained unclear, despite
extensive studies of their biosyntheses. Here we show that DKP
formation en route to the fungal virulence factor gliotoxin requires a
seemingly extraneous couplet of condensation (C) and thiolation (T)
domains in the NRPS GliP. In vivo truncation of GliP to remove the
CT couplet or just the T domain abrogated production of gliotoxin
and all other gli pathway metabolites. Point mutation of conserved
active sites in the C and T domains diminished cyclization activity of
GliP in vitro and abolished gliotoxin biosynthesis in vivo. Verified
NRPSs of other fungal DKPs terminate with similar CT domain
couplets, suggesting a conserved strategy for DKP biosynthesis by
fungal NRPSs.
Homology-based annotation of GliP uncovers two adenylation
(A), two condensation (C), and three thiolation (T) domains,
referred to as A1-T1-C1-A2-T2-C2-T3.[14] Previous work showed
that recombinant GliP converts L-Phe and L-Ser into the DKP
cyclo(L-Phe-L-Ser) (2), which was speculated to result from
spontaneous cyclization of
a GliP-T2-tethered-L-Phe-L-Ser
intermediate (Figure 1b).[7,14] Although 2 could plausibly be
derived from non-enzymatic cyclization of T2-tethered L-Phe-L-
Ser, the presence of the C2 and T3 domains, which in this model
have no function, may suggest an alternative mechanism. In
2012, Gao et al. reported that macrocyclization of linear peptidyl
precursors produced by a variety of fungal multi-module NRPSs
is catalyzed by conserved terminal condensation-like (CT)
domains,[15] and that CT domain activity was dependent on a
conserved histidine within the amino acid sequence
SHXXXDXXS/T. We noted that this CT-conserved sequence
exists in the GliP-C2 domain (Figure S1), suggesting that the C2
domain in GliP may be involved in cyclization of a tethered L-
Phe-L-Ser dipeptide (Figure 1b). We further hypothesized that
the seemingly extraneous T3 domain also plays a role in DKP
formation.
NRPS-derived DKPs form a large class of natural products with
diverse biological activities.[1-5] Gliotoxin (1) is the best-known
member of the epipolythiodiketopiperazines (ETPs), a family of
toxic DKPs produced by a variety of filamentous fungi.[1-5] The
biosynthesis of 1 has been extensively studied, due to its
significant contribution to the virulence of the devastating human
pathogen, A. fumigatus[6-12] and as a model system for fungal
NRPS pathways. Production of 1 is accomplished via the gli
biosynthetic gene cluster (BGC) in A. fumigatus and related
fungi (Figure 1a). The 13-gene gli BGC encodes the
transcriptional regulator, GliZ, one transporter (GliA), several
backbone tailoring enzymes, and the core NRPS, GliP (Figure
1a).[13] Whereas, the majority of gli-cluster tailoring enzymes
have previously been characterized extensively in vitro and in
[a]
[b]
Dr. J.A. Baccile, H.H. Le, C. Gomez, Prof Dr. F.C. Schroeder
Boyce Thompson Institute and Department of Chemistry and
Chemical Biology
Cornell University
Ithaca, New York, Unites States
E-mail: fs31@cornell.edu
B. T. Pfannenstiel, Dr. J.W. Bok, Prof. Dr. N.P. Keller
Departments of Bacteriology, Medical Microbiology and Immunology
University of Wisconsin-Madison
Figure 1. Gliotoxin biosynthesis in A. fumigatus. (a) gli-cluster gene
annotations, including characterized tailoring enzymes (blue), tailoring
enzymes with inferred functions (grey), and genes without predicted function
(black). gliZ: transcription factor; gliI: pyridoxal phosphate dependent
desulfurase; gliC, gliF: cytochrome P450 oxidases; gliM: O-methyltransferase;
gliG: glutathione-S-transferase; gliK: glutamate cyclase; gliA: transporter; gliN:
N-methyltransferase; gliT: oxidase; (b) Putative function of GliP and
abbreviated biosynthesis of 1 showing the most abundant intermediates or
shunt metabolites 2 and 3, as well as the detoxification product, 4.[6,9,16-19]
Madison, Wisconsin, United States
E-mail: npkeller@wisc.edu
[c]
E. Brandenburder, Prof. Dr. D. Hoffmeister
Department of Pharmaceutical Microbiology
Hans-Knöll-Institute, Friedrich Schiller University
Jena, Germany
Present Address: Division of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, CA, United States.
†
Supporting information for this article is given via a link at the end of
the document.
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Figure 2. LC-HRMS analysis of whole-metabolome extracts from WT and mutant A. fumigatus strains. (a-d) Representative overlaid extracted-ion-
chromatograms (EICs) for 1-4 in Af293 (black), GliP-ΔT3 (green), and GliP-ΔCTT3 (purple). Darker lines representative overlaid EICs for 1-4 in the indicated strains
with added exogenous 1. “10 x” indicates scaling of weak signals applied for comparison. “*” = unrelated peaks.
To investigate the function of the putative GliP-CT and the
GliP-T3 domains in vivo, we constructed two truncation mutants
in A. fumigatus, GliP-ΔT3 and GliP-ΔCTT3, and assessed the
impact of these truncations on the biosynthesis of 1 and gli-
pathway metabolites (1-4, Figure 2a-d). Comparison of whole-
metabolome extracts from WT (Af293), GliP-ΔT3, and GliP-
ΔCTT3 revealed a complete loss of 1-4 in the GliP-ΔCTT3 strain
(Figure 2a-d). In extracts from the GliP-ΔT3 strain we were
unable to detect 1 and the most abundant gli-pathway shunt
metabolites 2 and 3, however, we observed trace quantities of 4
(99 % less than Af293), the detoxification product of 1 (Figure
2a-d).[20] These data suggested that normal biosynthesis of 1
requires the T3 domain of GliP.
Gliotoxin (1) serves as a positive feedback loop for its own
production through regulation of gli-cluster expression.[21]
Accordingly, the inability of the GliP-ΔCTT3 mutant strain to
produce 1 resulted in almost complete loss of gli-cluster
expression (Figure S2), which could have affected our results.
Therefore, we repeated the experiment by growing cultures
supplemented with exogenous 1, which largely rescued gli-
cluster expression (Figure S3).[21] LC-HRMS comparison of
extracts from gliotoxin-supplemented cultures showed that
rescue of cluster gene expression did not recover production of
gli-pathway metabolites. As in the case without supplementation
of 1, the GliP-ΔCTT3 mutant did not produce any 1-3 (Figure 2a-
d), whereas the GliP-ΔT3 strain produced small quantities of 2
(96 % less than Af293) and the tailored metabolite, 3 (96 % less
than Af293) (Figure 2a-d). These results indicate that normal
biosynthesis of 1 requires the T3 domain of GliP, whereas in the
absence of T3, only small amounts of 1 and other gli-pathway
metabolites are produced, possibly via cyclization of a T2-
tethered intermediate.
expressed GliP truncations remained largely functional.
Production of cyclo-Phe-Ser (2) was only slightly reduced with
the GliP-ΔT3 mutant and still about one third of WT with the GliP-
ΔCTT3 mutant (Figure S6). Residual production of 2 by these
mutants likely results from spontaneous or CT-catalyzed
cyclization of the T2-tethered L-Phe-L-Ser (Figure 1b), which
does not appear to occur in vivo in the corresponding A.
fumigatus mutants (compare Figures 2a and S6).
Taken together, these results indicate that the GliP-T3
domain is required for DKP formation in vivo, since in the
absence of T3, only small amounts of 1 and other gli-pathway
metabolites are produced, possibly via cyclization of a T2-
tethered intermediate. Therefore, we hypothesized that in full
length GliP the L-Phe-L-Ser dipeptide is transferred from the T2
domain to T3 prior to cyclization via CT.
To assess whether the T3 domain is functional, we tested for
appropriate post-translational decoration of the predicted active
site residue, serine 2095, with a phosphopantetheinyl-(ppant-)
moiety. LC-HRMS/MS analysis of tryptic digests of GliP-WT
showed diagnostic ppant-fragmentation of the peptide containing
S2095, confirming ppant attachment to the active site serine in
T3 (Figure S7).[24]
To further probe the roles of the T3 and CT domains for DKP
formation, we constructed A. fumigatus strains carrying point
mutations at the T3 and CT active sites. For this purpose, we first
created a new ΔGliP strain, then reintroduced either GliP(WT), a
point mutant of the T3 active site, GliP-S2095A, or a point mutant
lacking the putative catalytic histidine of the CT domain, GliP-
H1754A (see Figure S2 and Supporting Methods for details).
The ΔGliP+GliP(WT) strain produced a distribution of pathway
metabolites (Figure 3a) very similar to that of WT A. fumigatus,
proving that the reintroduced GliP is fully functional. In contrast,
production of gli pathway metabolites was almost completely
abolished in the S2095A and H1754A mutant strains (Figure
3b,c). To rescue potentially suppressed expression of gli-cluster
genes in the absence of gliotoxin (1), we repeated the
experiment with exogenously added 1, which resulted in a
We further considered the possibility that the GliP-ΔCTT3 and
GliP-ΔT3 variants may have reduced adenylation activity and do
not load L-Phe and L-Ser as efficiently as the WT GliP. To
address this concern, we heterologously produced and purified
GliP-WT, GliP-ΔCTT3 and GliP-ΔT3 proteins from E. coli (Figure
S4) and conducted an ATP-[32P]pyrophosphate exchange assay. production of a small amount (<2% of WT) of cyclo-Phe-Ser in
We found that adenylation activity of the A1- and A2 –domains is
not reduced in the truncated proteins, GliP-ΔCTT3 and GliP-ΔT3,
relative to GliP-WT (Figure S5).[14,22] Notably, the heterologously
the S2095A mutant while cyclo-Phe-Ser remained undetectable
in the H1754A mutant (Figure 3b,c).
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Figure 3. LC-HRMS analysis of whole-metabolome extracts from mutant A. fumigatus strains. Representative EICs for compounds 1, 2 and 4 in (a) A. fumigatus
ΔGliP +GliP-WT, (b) A. fumigatus ΔGliP +GliP-S2095A, and (c) A. fumigatus ΔGliP +GliP-S2095A. Dashed chromatograms representative EICs for 1, 2, and 4 in
the indicated strains with added exogenous gliotoxin, 1. Note that the extent of conversion of gliotoxin (1) to its detoxification product 4 is highly variable between
experiments, e.g. in the example chromatogram shown in (a) no 1 remains following addition of 1.
To confirm the essential role of the GliP-T3 domain, we
heterologously expressed a GliP mutant protein in which the first
and second T-domains were inactivated by substitution of
serines 555 and 1582 for alanine (GliP-ΔT1T2). We then used
synthetic L-Phe-L-Ser-N-acetylcysteamine (L-Phe-L-Ser-SNAC,
5) to effect attachment of L-Phe-L-Ser to T3 via transthiolation
and monitored production of 2 (Figures 4a and S8a). Since
thioester 5 could cyclize non-enzymatically or via catalysis by
the CT domain without first getting attached to T3 (Figure S8b),
we included control assays without GliP, without Sfp, or without
coenzyme A (= no ppant functionalization on T3), and using a
GliP mutant protein in which all three T-domains were
inactivated (GliP-ΔT1T2T3). Incubation of
5
under these
conditions (Figure 4a) demonstrated that the presence of a
functional T3 domain significantly increases the cyclization
activity of GliP.
Our results demonstrate that cyclization of tethered Phe-Ser
dipeptide en route to 1 is not spontaneous, as proposed,[7-11] and
rather requires two additional GliP domains, the second
condensation-like domain (CT domain) and the enigmatic
terminal thiolation domain T3 (“TC” domain) to which the nascent
dipeptide appears to be transferred prior to cyclization via CT
(Figure 4b). Significantly, mutation of the catalytic histidine in
the CT domain or the ppant attachment site within the T3 domain
Figure 4.. (a) In vitro production of cyclo-Phe-Ser (2) using different GliP variants. Relative yields of 2 were measured via integration of the corresponding peak in
LC-MS ion-chromatograms (n = 4). **p < 0.01, ***p < 0.001. (b) Model for CTTC-catalyzed DKP formation in gliotoxin (1) biosynthesis.
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is sufficient to almost completely abrogate gliotoxin biosynthesis.
These observations highlight the importance of dedicated
cyclization domains in fungal NRPSs.[25,26] Gliotoxin (1) is a
representative member of a large class of NRPS-derived DKPs
that seem likely to be produced via similar mechanisms.
Analysis of available fungal genomes revealed 56 putative
NRPSs that feature terminal domains homologous to the CTT3
tandem in GliP (see Supporting Methods, Table S1, and
Figure S9), indicating a conserved biosynthetic strategy for DKP
formation.[13,27]
Whereas the GliP CT domain is homologous to the recently
described CT domains that catalyze cyclization of fungal
tripeptides;[15] the requirement of an additional TC domain for
DKP formation is perhaps unexpected, given that dipeptide
thioesters often cyclize non-enzymatically, as we showed in our
in vitro studies. We note that TC domains in DKP-producing
NRPSs could serve as a tether for linear dipeptides during
tailoring by other cluster enzymes. Although there is substantial
evidence that, in the case of gliotoxin, many of the later steps of
its biosynthesis proceed via untethered, cyclic intermediates, the
structures of shunt metabolites in related DKP biosynthesis
pathways may suggest tailoring of tethered dipeptides. For
example, in the case of hexadehydroastechrome, which is
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In conclusion, our study suggests a general framework for
fungal DKP biosynthesis, wherein the TC domain serves as a
tether for dipeptide cyclization by an adjacent CT domain and
potentially for tailoring by other cluster enzymes. Furthermore,
our characterization of the biosynthetic roles of the CT and TC
domains in DKP formation extends the functional repertoire of
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Acknowledgements
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This research was funded by an NIH Chemical Biology Interface
(CBI) Training Grant (5T32GM008500) to J.A.B., an NIH
Predoctoral
Training
Program
in
Genetics
Grant
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(5T32GM007133-40) to B.T.P., and NIH R01GM112739-01 to
N.P.K. and F.C.S. We thank Prof. Robert Cramer for the kind gift
of the GliP plasmid.
[25] K. D. Clevenger, R. Ye, J. W. Bok, P. M. Thomas, M. N. Islam, G. P.
Miley, M. T. Robey, C. Chen, K. Yang, M. Swyers, et al., ACS Chem.
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Conflict of interest
[26] M. T. Robey, R. Ye, J. W. Bok, K. D. Clevenger, M. N. Islam, C. Chen,
R. Gupta, M. Swyers, E. Wu, P. Gao, et al., Biochemistry 2018, 13,
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The authors declare no conflict of interest.
Keywords: gliotoxin • diketopiperazine • biosynthesis •
cyclization • NRPS
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Seemingly extraneous:
Joshua A. Baccile[a]†, Henry H. Le[a],
Brandon T. Pfannenstiel[b], Jin Woo
Bok[b], Christian Gomez[a], Eileen
Brandenburger[c], Dirk Hoffmeister[c],
Nancy P. Keller[b]*, Frank C.
Schroeder[a]*
Diketopiperazines derived from non-
ribosomal peptide synthetases, an
important family of fungal virulence
factors, do not form spontaneously, as
presumed; instead cyclization relies
on previously unannotated domains of
the synthetase.
Page No. – Page No.
Diketopiperazine Formation in Fungi
requires Dedicated Cyclization and
Thiolation Domains
e))
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