Opposed effects of enzymatic gliotoxin N - And S -methylations

Article abstract of DOI:10.1021/ja5033106

Gliotoxin (1), a virulence factor of the human pathogenic fungus Aspergillus fumigatus, is the prototype of epipoly(thiodioxopiperazine) (ETP) toxins. Here we report the discovery and functional analysis of two methyl transferases (MTs) that play crucial roles for ETP toxicity. Genome comparisons, knockouts, and in vitro enzyme studies identified a new S-adenosyl-l- methionine-dependent S-MT (TmtA) that is, surprisingly, encoded outside the gli gene cluster. We found that TmtA irreversibly inactivates ETP by S-alkylation and that this detoxification strategy appears to be not only limited to ETP producers. Furthermore, we unveiled that GliN functions as a freestanding amide N-MT. GliN-mediated amide methylation confers stability to ETP, damping the spontaneous formation of tri- and tetrasulfides. In addition, enzymatic N-alkylation constitutes the last step in gliotoxin biosynthesis and is a prerequisite for the cytotoxicity of the molecule. Thus, these specialized alkylating enzymes have dramatic and fully opposed effects: complete activation or inactivation of the toxin.

Full text of DOI:10.1021/ja5033106

Article
pubs.acs.org/JACS
Opposed Effects of Enzymatic Gliotoxin N- and S‑Methylations
Daniel H. Scharf,† Andreas Habel,† Thorsten Heinekamp, Axel A. Brakhage, and Christian Hertweck*
Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Beutenbergstrasse 11a, 07745 Jena,
Germany
Friedrich Schiller University, Furstengraben 1, 07737 Jena, Germany
̈
S
* Supporting Information
ABSTRACT: Gliotoxin (1), a virulence factor of the human
pathogenic fungus Aspergillus fumigatus, is the prototype of
epipoly(thiodioxopiperazine) (ETP) toxins. Here we report
the discovery and functional analysis of two methyl trans-
ferases (MTs) that play crucial roles for ETP toxicity. Genome
comparisons, knockouts, and in vitro enzyme studies identified
a new S-adenosyl-L-methionine-dependent S-MT (TmtA) that
is, surprisingly, encoded outside the gli gene cluster. We found
that TmtA irreversibly inactivates ETP by S-alkylation and that
this detoxification strategy appears to be not only limited to ETP producers. Furthermore, we unveiled that GliN functions as a
freestanding amide N-MT. GliN-mediated amide methylation confers stability to ETP, damping the spontaneous formation of
tri- and tetrasulfides. In addition, enzymatic N-alkylation constitutes the last step in gliotoxin biosynthesis and is a prerequisite for
the cytotoxicity of the molecule. Thus, these specialized alkylating enzymes have dramatic and fully opposed effects: complete
activation or inactivation of the toxin.
reactions play crucial, yet opposing, roles in gliotoxin
biosynthesis: ETP activation or irreversible inactivation.
INTRODUCTION
■
Pathogenic fungi produce a range of secondary metabolites to
weaken or kill the host.1,2 Structurally remarkable examples are
found in the heterogeneous family of epipoly-
(thiodioxopiperazine)s (ETPs) that are produced by a
phylogenetically diverse range of filamentous fungi.3 ETP
toxins are characterized by a diketopiperazine (DKP) core
furnished with unusual transannular di- or polysulfide
bridges,4,5 which are indispensable for their deleterious effects.
The epidithio residue enables ETPs not only to inactivate
proteins by conjugation to cysteine residues, but also to
generate reactive oxygen species through cycling between
oxidized (disulfide) and reduced (dithiol) forms.6 The
prototype of ETP toxins, gliotoxin (1; Figure 1), is an
infamous virulence factor of the human pathogenic fungus
Aspergillus fumigatus.7 It should be highlighted that invasive
aspergillosis caused by this fungus is a leading cause of death in
immunocompromised patients.8,9 Self-resistance of the pro-
ducer is, at least in part, conferred by the disulfide-forming
oxidoreductase GliT.10,11 Because of the eminent role of the
toxin, gliotoxin (gli) biosynthesis has been studied at the
synthetic,12 genetic, biochemical, and structural levels. In brief,
the DKP core is assembled by a nonribosomal peptide
synthetase (GliP),13−16 and the essential thiol residues are
introduced by sequential oxygenation (GliC) and glutathione
transfer (GliG),17,18 followed by a cascade of unusual
glutathione dissociation reactions catalyzed by GliK, GliJ,19
and GliI.20 Further enzymatic tailoring reactions such as
methylation have remained elusive. Here we report the
surprising finding that enzymatic N- and S-methylation
RESULTS AND DISCUSSION
■
Enzymatic Methylation Reactions in Gliotoxin Biosyn-
thesis. In the course of metabolic profiling of A. fumigatus, we
noted the formation of bis(methylthio)gliotoxin (2) as a minor
metabolite. This compound has been previously reported as a
derivative of gliotoxin in Trichoderma deliquescens (previously
Gliocladium deliquescens).21 Interestingly, S-methylation leads to
a complete loss of cytotoxic activity (Figure 1C). Moreover, in
contrast to the reversible detoxification by disulfide bond
formation, alkylation of the thiol moieties is irreversible. To
elucidate the biosynthetic origin of the methyl groups, we
performed stable isotope labeling experiments with 13C-
methionine. MS analyses of the A. fumigatus wild-type
(Af293) broth extract showed that all three methyl groups of
2 (M + 3; m/z 360 [M + H]+) and the N-methyl group of 1 (M
+ 1; m/z 328 [M + H]+) are methionine-derived and are thus
introduced enzymatically.
TmtA Catalyzes the S-Methylation of Gliotoxin. S-
Methyltransferases are scarce and have not yet been
characterized in fungi. The gliotoxin gene cluster of A.
fumigatus harbors two candidate genes, gliN and gliM, for
putative S-adenosyl-L-methionine (SAM)-dependent methyl-
transferases (MTs). A phylogenetic analysis showed that the
deduced gene products fall into distinct new clades in an MT
Received: April 2, 2014
Published: July 25, 2014
© 2014 American Chemical Society
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Figure 1. (A) Organization of the gli biosynthesis gene cluster. (B)
Structures and mass spectra of gliotoxin (1) and bis(methylthio)-
gliotoxin (2) compared to mass spectra of mono-13C-methylated 1 and
tri-13C-methylated 2 formed after incubation of the A. fumigatus wild
type (wt) with 13C-methionine. (C) Antiproliferative and cytotoxic
activities of 1 and its congeners (absolute values, Supporting
Information, Table S3).
Figure 2. (A) HPLC monitoring of the biotransformation reaction.
Extracted ion chromatograms are shown for (a) gliotoxin reference 1,
(b) incubation of 1 with the ΔgliM mutant, (c) incubation of 1 with
the ΔsmtA mutant, and (d) production of 1 by the ΔtmtA mutant. (B)
Detoxification reaction of 1 to 2 via 3 as an intracellular intermediate.
(C) Characterization and in vitro reconstitution of TmtA activity.
HPLC monitoring of the biotransformation reaction. Extracted ion
chromatograms are shown for (a) incubation of 3 in buffer showing
degradation to 1, 7, and 8, (b) incubation of 3 with heat-inactivated
TmtA only showing degradation to 7 and 8 (Figure 4), and (c) TmtA-
mediated transformation of 3 to 2. (D) Scheme of TmtA-mediated
biotransformation of 3 to 2. (E) Molecular mass of His6-tagged TmtA
analyzed by SDS−PAGE.
dendrogram (Supporting Information, Figures S1 and S2), yet
did not lend support for any candidate substrate. To investigate
the functions of the putative MT genes, we constructed
targeted A. fumigatus mutants lacking either gliN (ΔgliN) or
gliM (ΔgliM). We succeeded in generating the desired mutants
using a split-fragment polymerase chain reaction (PCR)-based
strategy.22 We verified successful gene deletion in both cases by
PCR and Southern blot analysis (Supporting Information,
Figure S4C). Notably, neither the ΔgliN mutant nor the ΔgliM
mutant produced gliotoxin. It is known that disruption of
essential gliotoxin biosynthesis genes, e.g., gliP, may result in
the down-regulation of the entire pathway. Moreover, gene
expression is typically restored by addition of gliotoxin,14
indicating an autoregulation of the gliZ transcription factor.
However, we could still detect 2 in ΔgliM (Figure 2A, trace b)
and ΔgliMΔgliN double-mutant (Figure S4B, trace b) cultures
treated with pure exogenous 1. To rule out the possibility of
redundant enzyme functions in A. fumigatus, we searched for
alternative S-MTs. Genome analysis led to the discovery of
another, albeit cryptic and partial, gliotoxin biosynthesis gene
cluster in A. fumigatus that consists of gene copies from gliC′,
gliZ′, gliP′, gliM′, and gliA′23 (Figure S4A). To test whether this
gliM-like gene is responsible for gliotoxin detoxification, we
constructed additional mutants lacking gliM′. Again, exogenous
gliotoxin was applied to cultures of A. fumigatus mutants ΔgliM′
and ΔgliMΔgliM′, yet LC/MS analysis still revealed formation
of 2 at a ratio similar to that of the single ΔgliM mutant (Figure
S4B). Therefore, we could rule out any contribution of GliN,
GliM, and GliM′ in gliotoxin detoxification by S-methylation.
Notably, gliM′ is not induced by addition of 1 as shown for the
gliotoxin cluster genes (Supporting Information, Figure S3).
We found that even Aspergillus nidulans and Fusarium
oxysporum, which lack any ETP biosynthesis gene cluster.24
are capable of ETP detoxification (Figure S4B). It thus appears
that S-methylation for detoxification is a trait not limited to
ETP producers and that the S-MT genes are not clustered with
ETP biosynthesis genes. Considering the high number of
putative MT genes present in the genomes of fungi, currently
an in vitro analysis of all candidates is out of the question.
However, comparative genome analyses of fungi known for
ETP S-methylation and extensive data mining yielded two
candidate MT genes, Afu4G13570 (S-methyltransferase, smtA)
and Afu2G11120 (thiol methyltransferase, tmtA), a homologue
of MT-II from Aspergillus niger (Figures S1 and S2) which is up-
regulated after exposure to dithiothreitol (DTT).25 Whereas
the ΔsmtA strain is still able to produce 2 (Figure 2A, trace c),
the cultures of the ΔtmtA strain lost the ability of S-methylation
and produced only 1 (Figure 2A, trace d). To verify the
proposed enzyme function, we performed in vitro experiments
using heterologously produced GliN, GliM, GliM′, SmtA, and
TmtA. Therefore, we amplified the corresponding open reading
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frames (ORFs), cloned the generated PCR fragments into
Escherichia coli expression vectors, and propagated the resulting
plasmids in E. coli DH5α cells. After sequencing of the insets,
the vectors were introduced into E. coli BL21(DE3) cells for
protein overproduction. After the cells were harvested, His6-
tagged GliN, GliM, GliM′, SmtA, and TmtA were purified
using a Ni Sepharose column. Gliotoxin (in its reduced form,
3) was individually incubated with the putative MTs in the
presence of SAM. Whereas no S-alkylation could be observed
for GliN, GliM, GliM′, and SmtA, the transformation of 1 into
2 could be observed only in the presence of TmtA (Figure 2C,
trace c).
carbon signal (at N2). Thus, 4 represents N-desmethylgliotox-
in, a novel intermediate in the gliotoxin pathway.
GliN Is a Monomeric, SAM-Dependent N-Methyltrans-
ferase. To verify the function of GliN as an amide N-MT, we
investigated the enzyme in vitro. Static light scattering
experiments revealed the homodimeric status of GliN, and
the theoretical molecular mass of a GliN monomer was found
to be 33.6 kDa, which is in excellent agreement with the
measured mass of 30.36 0.75 kDa (Figure 4C). Using SAM
When using heat-inactivated TmtA, S-methylation does not
take place. Taken together, we can rule out that the MT genes
in the gli gene locus contribute to S-methylation. Instead, this
reaction is carried out by a specialized thiol methyltransferase
(TmtA) encoded elsewhere in the genome.
Role of TmtA for Gliotoxin Resistance. To clarify the in
vivo function of TmtA, we performed an inhibition zone assay.
In contrast to the ΔgliT strain, which is deficient of the
resistance-conferring thiol−disulfide oxidoreductase, the tmtA
mutant shows only slightly increased sensitivity to 1 compared
to the wild type (Figure 3). A plausible explanation for this
Figure 3. Sensitivity of A. fumigatus wild-type (wt) and mutant strains
toward gliotoxin (1) (c = 4.5 mM) determined in an inhibition zone
assay.
observation is that TmtA serves as the second line of protection
against gliotoxin. In accordance with this, 2 is formed after
prolonged incubation times. It thus appears that TmtA does
not play the primary role in toxin self-resistance, but prevents
self-poisoning of the producing organism when exposed to the
toxin in higher titers or for longer periods. This model is also
supported by the kinetics of TmtA (vmax = 1.2 μmol min−1, Km
= 239.8 μM). Interestingly, detoxification by S-methylation was
also observed in bacteria such as the holomycin producer
Streptomyces clavuligerus; however, the S-MT in charge has
remained elusive.26 To our knowledge, microbial S-MTs that
modify natural products are unknown.
GliN Plays a Key Role in Gliotoxin Biosynthesis. The
surprising finding that S-alkylation is mediated by an enzyme
encoded outside the gli gene locus raised questions about the
roles of gliM and gliN. Thus, we more closely inspected the
metabolic profiles of the corresponding mutants. Whereas the
ΔgliM mutant did not show any new compound, the ΔgliN
mutant culture accumulated a slightly more polar compound
with m/z 313 [M + H]+. Traces of the same compound were
also detected in the wild type. Sufficient amounts of the new
compound for a full characterization were obtained from an
upscaled mutant culture. Purification by open column
chromatography on silica and preparative HPLC yielded pure
4 with a molecular formula of C12H13N2O4S2 as deduced from
HR-ESI-MS data (m/z 313.0312 [M + H]+, calcd for
C12H13N2O4S2 313.0311). 1D and 2D NMR data showed
that 4 has a spectrum similar to that of 1, yet lacking a methyl
Figure 4. Characterization and in vitro reconstitution of GliN activity.
(A) Southern blot analysis of gDNA of the wild type (wt) and the
ΔgliN mutants. (B) Molecular mass of His6-tagged GliN analyzed by
SDS−PAGE. (C) Native molecular size determination by static light
scattering. (D) HPLC monitoring of the biotransformation reaction.
Extracted ion chromatograms are shown for (a) incubation of 4 with
heat-inactivated GliN showing degradation to 5, (b) GliN-mediated
transformation of 4 to GT metabolites 1, 7, and 8, (c) GliN-mediated
transformation of 4 to GT metabolites 1, 7, and 8 in the presence of
GliT, and (d) the synthetic reference of 8. (E) Observed degradation
pattern for 4 followed by GliN-mediated biotransformation. (F)
Structure of N-desmethylgliotoxin (4) and key HMBC correlations.
as a cosubstrate, N-desmethylgliotoxin (4) was incubated with
GliN and heat-inactivated GliN, and the course of reaction was
monitored by LC/MS. Whereas no biotransformation of 4
could be detected with inactivated GliN (Figure 4D, trace a),
we observed that active GliN readily converted 4 into two new
products, 7 and 8 (Figure 4D, trace b), with m/z 359 [M + H]+
and m/z 391 [M + H]+, respectively. The increased [M + 2]+
peak and the Δm/z of 32 (64) compared to those of gliotoxin
indicated the formation of gliotoxin E27 and gliotoxin G,28
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Cryo platform. The chemical shifts are reported in parts per million
relative to the solvent residual peak (δ(CHCl3) = 7.26 ppm,
δ(CD3OD) = 3.31 ppm). The following abbreviations are used for
multiplicities of the resonance signals: s = singlet, d = doublet, t =
triplet, q = quartet, qt = quintet, br = broad. HPLC/MS was
conducted on a Thermo Surveyor Plus (LC) and Thermo LTQ Velos
(MS), an ESI source operating in positive mode. Preparative HPLC
purification was achieved by using a Gilson Abimed device with binary
pump 321 and UV/vis 156 (column, Phenomenex Luna C18, 10 μm,
250 × 21.2 mm; eluent, water, acetonitrile (20−100%)). HR-ESI-MS
was conducted on a Thermo Accela (LC) and Thermo Exactive
(HRMS), an ESI source operating in positive mode and an orbitrap
analyzer.
General Methods. All reagents were obtained from commercial
suppliers (Sigma-Aldrich, TCI, etc.) and used without further
purification unless otherwise explained. Reactions were carried out
under inert gas (argon) by using the Schlenk technique in dried
solvents. Open column chromatographic separations were executed on
silica gel (Kieselgel 60, 15−40 μm, Merck KGaA). Reaction progress
was monitored by thin-layer chromatography (TLC) (silica gel on
aluminum sheets (20 × 20 cm) with fluorescent dye, 254 nm, Merck
KGaA) or HPLC/MS.
General Strain Cultivation. Strains were grown in Aspergillus
minimal medium (AMM) as described previously.40 AMM agar was
prepared by addition of 1.6% (w/v) Select agar (Invitrogen,
Germany). For formation of conidia, A. fumigatus was cultivated on
AMM agar plates at 37 °C for 5 days. Conidia were harvested in 0.9%
(w/v) NaCl/0.1% (v/v) Tween 80 and counted using a CASY cell
counter (model TT, Innovatis AG, Germany).
Mutant Strain Cultivation in the Presence of Gliotoxin. All A.
fumigatus strains, ΔgliM, ΔgliM′, ΔgliMΔgliM′, ΔgliNΔgliM, ΔtmtA,
and ΔsmtA, were cultivated at 28 °C in 500 mL of Czapek Dox
medium at 180 rpm for 6 days. Gliotoxin (2 × 5 mg) was added on
days 2 and 3. Cultures of A. nidulans were handled equally. The
cultures were extracted twice with 300 mL of ethyl acetate. The
combined organic phases were dried over sodium sulfate and
concentrated to dryness. For gliotoxin and bis(methylthio)gliotoxin
determination, the residues were dissolved in 3 mL of MeOH and
measured with HPLC.
respectively. The latter was confirmed by comparison with a
synthetic reference.29 We found that the polysulfide formation
is reduced when GliT is included in the in vitro assay because
the enzyme promotes the formation of the transannular
disulfide bridge (Figure 4D, trace c). Furthermore, we observed
the rapid decomposition of 4 during NMR measurements.
Subsequent analysis of the decomposition products revealed
the presence of the S3- and S4-analogues of N-desmethylglio-
toxin, 5 (m/z 345.0033 [M + H]+, calcd for C12H13N2O4S3
345.0032) and 6 (m/z 376.9754 [M + H]+, calcd for
C12H13N2O4S4 376.9753). In stark contrast to that of gliotoxin,
which is stable over several hours, incubation of 4 leads to rapid
degradation within minutes. It is well conceivable that N-
methylation stabilizes ETP, and in fact free amide moieties are
exceptional for known ETPs.4,5 Most surprisingly, however, is
the finding that the loss of the amide methyl leads to a
complete loss of bioactivity: the antiproliferative and cytotoxic
potency of 4 is at least 100-fold reduced compared to that of
gliotoxin (Figure 1C).
A plausible explanation for this observation is that N-
methylation substantially contributes to the membrane
permeability of gliotoxin, as has been shown for other N-
methylated peptides.30 In addition, N-methylation might
influence the integrity of the sulfur bridge as shown for the
pentathiepin heterocycle.31 From a biosynthetic viewpoint, it is
noteworthy that GliN is uniquely positioned among enzymes
that methylate amide nitrogens. Although about 23% of all
known MTs are classified as N-MTs,32,33 most of the well-
characterized examples catalyze the methylation of amine
nitrogens. Known amide N-MTs are either of bacterial origin or
partial domains of larger enzymes.34−36 Furthermore, the
substrate specificity of GliN is unique among known fungal
MTs.37
CONCLUSION
■
Cultivation of the Wild Type in the Presence of 13C-
Methionine. A. fumigatus strain CEA17ΔakuB was cultivated at 28
°C in 500 mL of Czapek Dox medium at 180 rpm for 6 days. 13C-
Methionine (2 × 25 mg) was added on days 2 and 3. The cultures
were extracted twice with 300 mL of ethyl acetate. The combined
organic phases were dried over sodium sulfate and concentrated to
dryness.
Isolation and Manipulation of Nucleic Acids. Isolation of
chromosomal DNA of A. fumigatus was done using the Master Pure
Yeast DNA Purification Kit (Epicentre Biotechnologies, United
States). For Southern blot analysis, DNA fragments were separated
on an agarose gel and blotted onto Hybond N+ nylon membranes
(GE Healthcare Life-Sciences, Germany). Labeling of DNA probes,
hybridization, and detection of DNA−DNA hybrids were performed
as described previously.41
Generation of A. fumigatus Mutant Strains. Partial deletion of
gliN was achieved by a PCR-based strategy. Upstream and downstream
flanking regions of gene gliN (AFUA_6G09720) were amplified by
PCR using primer pairs gliN5-for and gliN-ptrA-rev and gliN3-rev and
gliN-ptrA-for, respectively. By this reaction, overlapping ends of the
pyrithiamine resistance cassette were introduced at the 3′-end of the
upstream flanking region and at the 5′-end of the downstream flanking
region of the gliN gene. The ptrA resistance cassette was amplified
from plasmid pSK275 with primers ptrA-for and ptrA-rev. All PCR
fragments were purified by gel extraction. The final deletion construct
was generated by a three-fragment PCR employing primers gliN5-for
and gliN3-rev. All PCRs were performed with Phusion high-fidelity
DNA polymerase (Finnzymes) according to the manufacturer’s
recommendations. The resulting 3.5 kb PCR product was purified
and used for transformation of A. fumigatus wild-type protoplasts.
Pyrithiamine (1 mg mL−1) resistant transformants were analyzed for
In summary, we have elucidated the roles of two new MTs that
are crucially associated with gliotoxin tailoring. We found that
enzymatic S-methylation is a strategy of ETP producers and
some nonproducers to irreversibly inactivate ETP toxins. This
reaction is catalyzed by TmtA, the first example of an ETP S-
MT. In general, there are only a few examples of S-MTs from
plants and animals,38,39 and microbial S-MTs are unprece-
dented. Surprisingly, these S-MTs are not encoded in the ETP
biosynthesis gene clusters, which hampered their discovery.
From a biotechnological point of view, ΔtmtA strains may be
employed as valuable sources of ETPs, preventing compound
losses by S-alkylation. In this study, gene inactivation, isolation
of a new pathway intermediate (4), and the successful in vitro
biotransformation of 4 into 1 also elucidated the last step in
gliotoxin biosynthesis. The SAM-dependent N-methylation of
the diketopiperazine core not only confers stability to the
molecule, but also turns a harmless precursor into a potent
toxin. Thus, N- and S-methylations of gliotoxin represent an
unusual case of turning on and off the toxicity of a virulence
factor by simple, yet regiospecific and substrate-specific,
methylation. Without doubt, this finding will have implications
for related natural products of the large family of ETP toxins.
EXPERIMENTAL SECTION
■
General Analytical Procedures. All 1D (1H, 13C, DEPT) and 2D
(1H−1H COSY, HSQC, NOESY, HMBC) NMR spectra were
recorded in deuterated solvents on a Bruker AVANCE II 300 MHz
or AVANCE III 500 or 600 MHz instrument equipped with a Bruker
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partial deletion of gliN by Southern blot analysis. One positive
transformant, designated ΔgliN, was chosen for further analysis. The
gene gliM (AFUA_6G09680) was likewise deleted using primer pairs
gliM5-for and gliM-ptrA-rev and gliM3-rev and gliM-ptrA-for. The
resulting 3.6 kb PCR fragment was used for transformation. The gene
tmtA (AFUA_2G11120) was likewise deleted using primer pairs
tmtA5-for and tmtA-ptrA-rev and tmtA3-rev and tmtA-ptrA-for. The
resulting 3.9 kb PCR fragment was used for transformation. The gene
rate, 200 μL min−1; 0.1% formic acid in water (solvent A), 0.1% formic
acid in acetonitrile (solvent B); gradient, 1 min 5% B, 1−16 min 5−
98% B, 16−19 min 98% B, 20−28 min 5% B.
In Vitro Biotransformation Assay Using GliM, GliM′, SmtA,
and TmtA. The enzymatic activity of purified proteins was measured
by incubation of reduced gliotoxin with pure protein or heat-
inactivated protein as the control for 2 h and S-adenosylmethionine as
a cosubstrate. After EtOAc extraction (2 × 250 μL) the organic solvent
was dried under a stream of nitrogen, and the residues were
redissolved in 100 μL of methanol and analyzed by HPLC/MS.
HPLC conditions: column, Thermo Betasil (C18, 150 × 2.1 mm, 5
μm); flow rate, 200 μL min−1; 0.1% formic acid in water (solvent A),
0.1% formic acid in acetonitrile (solvent B); gradient, 1 min 5% B, 1−
16 min 5−98% B, 16−19 min 98% B, 20−28 min 5% B. The kinetic
characterization of TmtA was carried out with the SAM
methyltransferase assay (G-Biosciences). The concentrations of
protein (1 μM) and SAM (200 μM) were kept constant, while the
concentration of reduced gliotoxin was varied.
̀
gliM (Afu3g12910) was likewise deleted using primer pairs gliMlike5-
for and gliMlike-hph-rev and gliMlike3-rev and gliMlike-hph-for. The
hygromycin resistance cassette was amplified from plasmid pUChph
with primers hph-for and hph-rev. The resulting 4.8 kb PCR fragment
was used for transformation. Hygromycin (250 μg mL−1, InvivoGen,
France) resistant transformants were analyzed for partial deletion of
̀
gliM by Southern blot analysis. The gene smtA (Afu4G13570) was
likewise deleted using primer pairs smtA5-for and smtA-hph-rev and
smtA3-rev and smtA-hph-for. The resulting 5 kb PCR fragment was
used for transformation.
Isolation of Gliotoxin from A. fumigatus. A. fumigatus was
cultivated in Czapek Dox medium for 5 days (28 °C, 180 rpm). The
broth was filtered (pleated filters), the mycelium was washed with
water, and the combined culture filtrate was extracted twice with half
its volume of chloroform. The crude extract was resuspended in a
minimal volume of methanol and extracted three times with hexane to
remove all lipids. The methanol fraction was evaporated and the crude
material resuspended in ethanol, treated with charcoal, and stored at 2
°C overnight for crystallization. The crystallized gliotoxin was filtered
and recrystallized from ethanol.
Synthesis of Gliotoxin G Reference. Synthesis was performed
according to a published method.29 Briefly, to a solution of gliotoxin 6
(10 mg, 31 μmol) and rhombic sulfur (100 mg) (recrystallized from
toluene) in carbon disulfide (1 mL) was added lithium phenyl-
methanethiolate (3.1 μmol, 0.1 equiv) (prepared from benzenethiol
and nBuLi in THF) at room temperature. After being stirred for 15 h,
the suspension was poured onto a silica plug, which was eluted by
dichloromethane to remove excess sulfur, followed by ethyl acetate.
The crude fraction was subjected to preparative HPLC. HPLC
conditions: flow rate, 21 mL min−1; water (solvent A), acetonitrile
(solvent B); gradient, 1 min 10% B, 1−18 min 10−100% B, 18−20
min 100% B, 20−24 min 10% B. Compound 8 eluted after 15.1 min.
The yield was 4.8 mg (48%) as a white solid. The spectroscopic data
agreed fully with those reported.
Heterologous Production and Purification of Proteins. The
gliN ORF (849 bp) was amplified by PCR using primer pair gliN
forward and gliN reverse. The generated PCR fragment was cloned
into the BamHI/HindIII sites of pET43.1H6GliT to create
pET43.1H6GliN. The resulting plasmid was propagated in E. coli
DH5α cells. The DNA sequence was verified by sequencing, and the
vector was introduced into E. coli BL21(DE3) cells for protein
overproduction. Recombinant GliN was produced by autoinduction in
Overnight Express instant TB medium (Novagen). Cells were
harvested after 24 h of incubation at 30 °C. His6-GliN-containing
cleared cellular extracts in 20 mM Tris/HCl, 150 mM NaCl, pH 7.5,
were applied to a 25 mL Ni Sepharose 6 FF column (all columns
purchased from GE Healthcare Bio-Sciences, Freiburg, Germany).
GliN was eluted with 200 mM imidazole. GliN was applied to a
HiLoad 26/60 Superdex 200 pg column. The GliN-containing fraction
was identified by SDS−PAGE and stored in 50% glycerol at −20 °C.
Protein concentrations were determined according to Bradford using
the CoomassiePlus protein assay reagent (Pierce). The gliM ORF
(1257 bp) was amplified by PCR using primer pair gliM forward and
gliM reverse. The generated PCR fragment was cloned into the
BamHI/NotI sites of pET43.1H6GliT to create pET43.1H6GliM.
Protein production and purification of GliM were similar to GliN
purification. All other proteins were cloned and purified as described
for GliN.
Reduction of Gliotoxin. Gliotoxin (20 mg, 61.3 μmol) was
suspended in 2-propanol (2 mL), cooled to 0 °C, and treated
successively with NaBH4 (18.5 mg, 0.49 mmol, 8 equiv). After being
stirred for 30 min at 0 °C, the reaction mixture was poured into 1 M
HCl (20 mL) and extracted twice with EtOAc (20 mL). The
combined EtOAc fractions were dried over Na2SO4 and evaporated,
yielding bis(methylthio)gliotoxin (18.2 mg, 90.4%).
Inhibition Zone Assays for Gliotoxin Sensitivity. The
sensitivity of various A. fumigatus strains to gliotoxin was determined
in an inhibition zone assay. Of the test strains, 2 × 107 conidia were
mixed with 20 mL of YAG agar (0.5% (w/v) yeast extract, 2% (w/v)
agar, 2% (w/v) glucose, trace elements). Next, 100 μL of 4.5 mM
gliotoxin was added in a hole in the center of the plate, and the
diameter of the inhibition zone was determined after 24 h of
incubation at 37 °C. The experiment was repeated in triplicate.
Determination of Physical and Biochemical Properties of
GliN. The absolute molecular mass was determined by static light
scattering experiments using a miniDawn Treos monitor (Wyatt).
Therefore, pure GliN was applied to a HiLoad 16/60 Superdex 200 pg
column (GE Healthcare), and identified peaks were processed by
ASTRA 6 software (Wyatt). The absolute molecular mass of native
GliN was calculated from both the UV and light scattering responses.
Isolation of Desmethylgliotoxin from the ΔgliN Mutant. The
ΔgliN mutant was cultivated in 2 L (4 × 500 mL) of Czapek Dox
medium in a 2 L Erlenmeyer flask for 5 days (28 °C, 220 rpm). The
broth was filtered (pleated filters), the mycelium was washed with
water, and the combined culture filtrate (2 L) was extracted twice with
a half volume of EtOAc. The crude extract of the ΔgliN mutant was
subjected to silica gel chromatography using cyclohexane/EtOAc
(starting from 80:20 with increasing amount of EtOAc). Fractions
containing 4 were combined and further purified by preparative
HPLC. HPLC conditions: column, Macherey Nagel (C18 Nucleodur
Polartec, 250 × 10 mm, 5 μm); flow rate, 4.8 mL min−1; water
(solvent A), acetonitrile (solvent B); gradient, 1 min 10% B, 1−18 min
10−100% B, 18−20 min 100% B, 20−24 min 10% B. Compound 4
eluted after 12.0 min. The yield was 4.2 mg.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed phylogenetic tree, Northern blot, accession numbers,
originating species, and function of the proteins used for
phylogenetic analysis, oligonucleotides used in this study,
acitivity testing of 1, 2, and 4, physiochemical data for 4, full
spectra for 4, HR-ESI-MS spectra of 5 and 6, kinetic analysis of
S-methylation by TmtA, and UV and MS traces from the S-
methylation assay. This material is available free of charge via
the Internet at http://pubs.acs.org.
In Vitro Biotransformation Assay Using GliN. The enzymatic
activity of purified GliN was measured by incubation of 3 with GliN or
heat-inactivated GliN as the control for 2 h and S-adenosylmethionine
as a cosubstrate. After EtOAc extraction (2 × 500 μL) the organic
solvent was dried under a stream of nitrogen, and the residues were
redissolved in 100 μL of methanol and analyzed by HPLC/MS. HPLC
conditions: column, Thermo Betasil (C18, 150 × 2.1 mm, 5 μm); flow
11678
dx.doi.org/10.1021/ja5033106 | J. Am. Chem. Soc. 2014, 136, 11674−11679

Journal of the American Chemical Society
Article
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AUTHOR INFORMATION
Corresponding Author
Christian.Hertweck@hki-jena.de
■
Author Contributions
†D.H.S. and A.H. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank A. Perner for MS measurements, S. Fricke and C.
Schult for technical assistance, and Dr. H. M. Dahse for
cytotoxicity screening. Financial support by the DFG is
gratefully acknowledged.
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