Sequential Inactivation of Gliotoxin by the S-Methyltransferase TmtA

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Sequential inactivation of gliotoxin by the S-methyltransferase TmtA Elke R. Duell, Manuel Glaser, Camille Le Chapelain, Iris Antes, Michael Groll, and Eva M. Huber ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00905 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on February 1, 2016

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ACS Chemical Biology

Sequential inactivation of gliotoxin by the S-methyltransferase TmtA Elke R. Duell,‡ Manuel Glaser,‡ Camille Le Chapelain, Iris Antes, Michael Groll and Eva M. Huber* Center for Integrated Protein Science Munich (CIPSM) at the Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany ABSTRACT: The epipolythiodioxopiperazine (ETP) gliotoxin mediates toxicity via its reactive thiol groups and thereby contributes to virulence of the human pathogenic fungus Aspergillus fumigatus. Self-intoxication of the mold is prevented either by reversible oxidation of reduced gliotoxin, or by irreversible conversion to bis(methylthio)gliotoxin. The latter is produced by the S-methyl transferase TmtA and attenuates ETP biosynthesis. Here we report the crystal structure of TmtA in complex with S-(5’adenosyl)-L-homocysteine. TmtA features one substrate and one cofactor binding pocket per protein and thus, bisthiomethylation of gliotoxin occurs sequentially. Molecular docking of substrates and products into the active site of TmtA reveals that gliotoxin forms specific interactions with the protein surroundings and free energy calculations indicate that methylation of the C10a-SH group precedes alkylation of the C3-SH-site. Altogether, TmtA is well suited to selectively convert gliotoxin and to control its biosynthesis, suggesting that homologous enzymes serve to regulate the production of their toxic natural sulfur compounds in a similar manner.

The opportunistic fungus Aspergillus fumigatus causes in1 vasive aspergillosis in immunocompromised patients. The secondary metabolite gliotoxin largely contributes to the 2, 3 virulence of this pathogen. Chemically, gliotoxin belongs to the class of epipolythiodioxopiperazine (ETP) compounds. 4 Its thiol groups are essential for bioactivity, as they conjugate to sulfur-containing proteins, disturb the intracellular redox equilibrium and generate reactive oxygen species by 5 cycling between reduced and oxidized states. Gliotoxin is synthesized by an enzymatic cascade encoded in the gli biosynthesis gene cluster. Starting from the proteinogenic amino acids L-Phe and L-Ser the non-ribosomal peptide synthe6 tase GliP produces the diketopiperazine core that is further oxidized and covalently linked to two glutathione (GSH) 7 molecules. Sequential degradation of the glutathione moie8 ties creates the toxic dithiol form of gliotoxin and the flavin adenine dinucleotide (FAD)-dependent oxidoreductase GliT finally installs the disulfide bridge to prevent self-poisoning 9-11 of the fungus (Figure 1a). For many ETPs like gliotoxin, a biologically inactive bis12-14 thiomethylated derivative is generated as well (Figure 1b). Recently, the enzyme converting the dithiol form of gliotoxin to bis(methylthio)gliotoxin has been identified. Surprisingly, the S-methyltransferase TmtA (or GtmA) is encoded outside the gli gene cluster, though its expression is inducible by 15, 16 gliotoxin. TmtA utilizes the cofactor S-(5’-adenosyl)-Lmethionine (SAM) to irreversibly methylate the sulfhydryl groups of reduced gliotoxin, thereby eliminating its toxicity 17, 18 (Figure 1a). By displaying a lower affinity for reduced gliotoxin than GliT, TmtA plays a minor role for the self15, 16 resistance of A. fumigatus. However, the bis-

thiomethylated compound was shown to negatively regulate gliotoxin biosynthesis and hence to prevent uncontrolled 16 Recently, bisproduction of the natural product. thiomethylated gliotoxin has been suggested as a diagnosis 19, 20 marker of invasive aspergillosis. This clinical relevance of TmtA, the significance of TmtA inactivation for loss-free biotechnological production of ETPs, TmtA’s low sequence homology (< 20%) to structurally characterized methyltransferases (MTs; Supporting Information Figure S1a, b), and the rare use of sulfur as the target atom for enzymatic methyla21 tion (3% of all MTs) prompted us to analyze the mode of action of this S-MT at atomic resolution. RESULTS AND DISCUSSION TmtA was heterologously expressed in Escherichia coli, purified (Supporting Information Figure S2a,d), and crystallized. Native diffraction data were collected to 1.5 Å resolution and the structure was phased by single-wavelength anomalous dispersion using selenomethionine labelling (PDB ID 5EGP, Rfree 16.4%, Supporting Information Table S1). The asymmetric unit contains two TmtA copies, which are structurally identical (root-mean-square deviation (r.m.s.d.): 0.178 Å). They form a dimeric assembly with two independ2 ent active sites and an interface area of 1285.5 Å (Supporting Information Figure S3). Even though the homodimer is considered a stable quaternary structure, size exclusion chromatography under non-reducing conditions supposes a monomeric state for TmtA in solution (Supporting Information Figure S2a, b). Therefore dimerization looks like to be a crystallization artefact. TmtA represents a stable single

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Figure 1. Inactivation of gliotoxin by methylation or oxidation. a) GliT (left) oxidizes the free thiols of gliotoxin at the positions 3 and 10a to a disulfide-bridge. By contrast, TmtA (right) irreversibly inactivates reduced gliotoxin by S-methylation. b) Chemical structures of bis-thiomethylated ETPs. domain protein (TM= 55 °C, Supporting Information Figure S2e) that adopts the Rossmann-like fold, a hallmark of SAMdependent MTs. The central β-sheet, which contains seven th strands with the 7 running antiparallel to all others, is 22 flanked by α-helices on both sides (Figure 2). The two Nterminal helices and helical inserts between the sheets 6 and 7 classify TmtA as a small-molecule MT (Supporting Infor22 mation Figure S1c). TmtA displays significant 3D similarity to a variety of hypothetical MTs. Its closest characterized relative is the yeast C-methyltransferase Coq5 (r.m.s.d. 2.7 Å; Supporting Information Figure S4and Table S2). Even though SAM was added to the protein prior to crystallization, SAH was unambiguously identified in the 2FO-FC electron density map (Figure 2). Most likely, the loosely bound, surface-exposed methyl group of SAM dissociated from the coenzyme’s sulfur atom during irradiation of the crystal. The active site of TmtA contains only one SAH molecule, suggesting that conversion of gliotoxin to its bisthiomethylated form is catalyzed sequentially with generating mono-alkylated species as intermediates. In agreement, under conditions where SAM is limiting, mono(methylthio)16 gliotoxin was detected in vitro. The conformation of SAH in the active site of TmtA is similar to that of other MTs (Supporting Information Figure S5). The amino group of the adenine moiety interacts with Asn109OD1 (3.2 Å) and the hydroxyl groups of the ribose are hydrogen-bonded to Asp82OD1/2 (2.7 Å). This stabilization of the sugar moiety is 23 common to all SAM-dependant MTs. Tyr20OH as well as Thr27OH interact with the carboxylate of the methionine moiety, while the main chain atoms Ala54O and Ala126O coordinate the N-terminus of the methionine residue (2.8 Å; Figure 2). Ala54O is part of an AxGxG motif (Supporting Information Figure S1a), which is related to the GxGxG sequence typical of nucleotide binding sites. Altogether, these features classify TmtA as a class I methyltransferase. Assuming that SAH and SAM adopt the same conformation (Supporting Information Figure S5), the donor methyl group of the cosubstrate points into the substrate binding cleft and

undergoes weak hydrophobic interactions with Phe11CE1 (4.4 Å) and Phe127CB (4.1 Å). Our crystallographic data revealed a well-shaped hydrophobic cavity close to the cofactor, which most likely represents the substrate docking pocket (Supporting Information Figure S6a). It is formed by residues of the N-terminus, helices H1, H7, H8, and H10, sheet S5, as well as the loop region connecting sheet S4 and helix H6. The residues Met10, Phe11, Ser131, His189, Tyr237, and Lys241 restrict access to the active site. Since crystal soaking experiments and cocrystallization trials with reduced gliotoxin and bis(methylthio)gliotoxin in the presence of either SAH or SAM failed, we performed computational studies using the FlexX-Pharm molecular docking method. For the best scored protein:ligand complexes molecular dynamics simulations were conducted and free energy estimates for ligand binding were calculated via the MM-PBSA and MM-GBSA approaches (for details see Supporting Information). The best scoring conformation for reduced gliotoxin (Figure 3a and Supporting Information Table S3) fits well into the substrate binding cleft (Supporting Information Figure S6b). The hydrophobic parts of the pyrrolidine and phenylalanine-derived ring moieties undergo apolar contacts with the residues Met10CE, Phe11CZ, Phe185CE2, Leu188CD1 and His189CB (3.8–4.8 Å), while the C3-SH group attached to the diketopiperazine core is stabilized by the residues Leu26CD2, Thr27CG2, Trp157CZ2 and Trp162CZ2 (3.5–4.5 Å; Figure 3a and Supporting Information Figure S7). Furthermore, Trp162NE1 interacts with the serine side chain of gliotoxin (2.9 Å) and Tyr20OH binds to the carbonyl group of the diketopiperazine moiety (3.0 Å). The hydroxyl group attached to the phenylalanine moiety of gliotoxin is in hydrogen bonding distance to the main chain oxygen of Phe185 (2.7 Å; Figure 3a). In addition, Thr23CG2, Leu26CD2 and Met233CE mediate hydrophobic contacts with the N-methyl group of gliotoxin (3.1–4.7 Å). From these structural data we infer that TmtA is highly specific for reduced gliotoxin, although its KM value is quite high (240 15 µM). In support of this conclusion TmtA does not convert


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Figure 2. Crystal structure of TmtA. Left: Ribbon illustration of TmtA with the cofactor SAH (green) shown as ball-and-stick model. Right: Close-up of the SAH binding site. The amino acids engaged in cofactor binding are depicted as sticks and labelled by the one-letter-code. Hydrogen bonds are depicted as dashed black lines. The 2FO-FC omit electron density map for SAH (blue mesh) is contoured at 1σ. 16

the ETP sporidesmin. To validate the modeling results, we created two TmtA mutants, Y20A and W162A. Both amino acids form hydrogen bonds to SAM and/or gliotoxin and are conserved in TmtA homologues from various fungi (Figures 2 and 3 as well as Supporting Information Figure S1b). Activity assays with WT and mutant TmtA showed only for the WT enzyme reasonable reaction kinetics (Supporting Information Figure S8). Table 1. Relative binding free energies (∆∆G) of gliotoxin ligands to cofactor bound TmtA and of the coenzymes to apo-TmtA (see also Table S3). Reaction step

∆∆GFlexX-Score [kcal mol-1]a

∆∆GMM-PBSA [kcal mol-1]

∆∆GMM-GBSA [kcal mol-1]

C10a-C3 S-methylation sequence (cf. Figure 3) 1 b

2 3

c

4b,c

1.80

–4.66

–7.02

–0.97

–11.90

–0.24

0.74

–9.29

–6.21

–2.37

–8.37

–9.31

C3-C10a S-methylation sequence (cf. Supporting Figure S9) 1 b

2

1.41

3.34

5.26

–0.30

–22.95

–17.11

c

0.46

–6.24

–1.62

4b,c

–2.37

–8.37

–9.31

–17.13

–11.39

3

Coenzyme exchange SAH-SAM a

–0.40

The estimates for ΔΔG obtained by the FlexX docking score are all close to zero. Although this is in agreement with the low KM value,15 the differences are smaller than the overall accuracy of docking scoring functions (~ 2 kcal/mol) and thus not significant. We therefore performed additional MM-PBSA and MM-GBSA molecular dynamics calculations based on the FlexX-Pharm docking poses (columns 2 and 3). bThe coenzyme exchange free energy was added to the gliotoxin ligand free energy of binding. cThe ∆∆G values are based on the free energies of binding of bis(methylthio)gliotoxin in pose 2. Abbreviations: MM-PBSA: Molecular Mechanics – Poisson Boltzmann Surface Area, MM-GBSA: Molecular Mechanics – Generalized Born Surface Area.

According to the modeling data, the C10a-sulfhydryl group of reduced gliotoxin is positioned adjacent to the reactive methyl group of SAM (3.3 Å), while the C3-thiol is located far more distant from the cofactor (5.9 Å, Figure 3a). Since the SN2-like nucleophilic substitution reaction mechanism of MTs requires close proximity of the reactants, the C10a-sulfur atom is likely to be methylated first (Figure 3b). Notably, the active site of TmtA does not contain any basic amino acids that could specifically initiate the reaction by deprotonation of the nucleophilic thiol group as it was described e.g. for 24, 25 Docking of the monomethylated gliotoxin species Coq5. revealed that stepwise alkylation of first the C3- and second the C10a-SH-group would trigger significant conformational and energetically disfavored changes of the ligand in the substrate binding pocket (Supporting Information Figure S9). In contrast, initial thiomethylation at the C10a-SH group followed by C3-SH alkylation requires only minor structural rearrangements of gliotoxin and the intermediate would be well stabilized by hydrophobic and polar contacts similar to the unmodified educt (Figure 3a, b). Favorable van der Waals contacts of the C10a-thioether with Met10, Phe11, Phe16, Tyr20, Met233 and the sulfur atom of SAH (3.7–4.5 Å) cause the ligand to twist by ~50° (Figure 3b, e). For steric reasons, exchange of the consumed cofactor (SAH) by a new molecule SAM shifts the C10a-thioether further into this hydrophobic cleft and perfectly positions the C3-sulfur atom for the second methylation (distance to the CH3-group of SAM: 3.9 Å; Figure 3c and e). Support for this reaction trajectory (C10aC3; Figure 4) is provided by molecular dynamics simulations and MD-based MM-PBSA and MM-GBSA calculations of the changes in binding free energies ΔΔG (Table 1). ΔΔG is negative, i.e. favorable, for initial C10a-methylation (– 4.66/–7.02 kcal/mol) but not for primary alkylation of the C3 position (3.34/5.25 kcal/mol; Table 1). In agreement, C3methylation in the second reaction step is energetically more favorable than modification of the C10a-SH group (C3: – 9.29/–6.21 versus C10: –6.24/–1.62 kcal/mol). Furthermore, the ΔΔG values and the absolute binding free energies associated with the SAH to SAM exchange are much larger (– 17.13/–11.39 kcal/mol) than the values associated with changes in gliotoxin ligand binding (Table 1 and Supporting Information Table S3). These calculations imply that exchange of



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Figure 3. Molecular docking of gliotoxin ligands into TmtA. The protein is colored in gray; for clarity the residues 126–132, 155– 160, 187–193 and 273–281 are not displayed; side chains mediating important contacts to ligands are shown as sticks and labelled by the one-letter amino acid code. Hydrogen bonds and hydrophobic contacts are indicated by dotted black lines. a) Reduced red gliotoxin (GT , magenta) is well stabilized in the active site. The C10a thiol group is located close to the reactive methyl group of SAM (black arrow; 3.3 Å). b) Upon methylation of the C10a-SH-group, SAH is produced and the modified natural product mm (GT , lightblue) shifts in the active site to position the C3-SH group for the second methyl transfer. c) Exchange of SAH by a new SAM molecule enables methylation of the C3-SH group of gliotoxin (distance 3.9 Å). d) The final reaction product, bm red bis(methylthio)gliotoxin (pose2, GT , orange), is formed. e) Structural superpositions of reduced (GT , magenta), monomm bm (GT , lightblue/gray) and bis-thiomethylated gliotoxin (GT , orange) illustrate conformational changes during the C10aC3 methylation sequence. the gliotoxin intermediate is energetically less favorable and slower than exchange of the cofactor, which thus might take place “on the fly” during the reaction cycle. As a consequence, the second methylation occurs before the monoalkylated intermediate is released from the enzyme’s active site. Docking of the final product bis(methylthio)gliotoxin

resulted in two distinct poses with an almost equal docking score (Supporting Information Figures S9, S10 and Table S3). Conformation 2 matches well that of reduced and C10amono-thiomethylated gliotoxin (Figure 3d), while pose 1 is significantly shifted in the active site and therefore was considered to be unlikely (Supporting Information Figure S10).


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Figure 4. Proposed methylation sequence of reduced gliotoxin by TmtA. Modelling data suggest that conversion of reduced gliotoxin to bis(methylthio)gliotoxin occurs via the C10a-mono-thiomethylated intermediate. Mono-thiomethylation at the C3SH group is less likely (gray route). The structural flexibility of the product might also reflect the attempt to get released from the enzyme and explain why it could not be trapped in a defined orientation in the TmtA crystal structure. To further validate our modeling results and to investigate the influence of the aforementioned nonpolar pocket formed by Met10, Phe11, Phe16 and Tyr20 on ligand binding, we created in silico the quadruple mutant M10A, F11A, F16A, Y20A. Strikingly, docking studies with gliotoxin ligands in the presence of either SAH or SAM yielded neither poses for C10amono-thiomethylated nor for bis-thiomethylated gliotoxin, whereas docking of reduced gliotoxin to the SAM-bound TmtA mutant and docking of the C3-mono-thiomethylated gliotoxin species to the SAH-bound TmtA variant were successful. These data emphasize that this hydrophobic pocket is essential for binding of the C10a-mono-thiomethylated gliotoxin species. Regarding the calculated energetics, both the FlexX scores as well as the absolute values of the MMPBSA/MM-GBSA estimated free energies of binding are more favorable for the wild-type (WT) than for the mutant (MT) protein, suggesting that the WT residues have a stabilizing effect on gliotoxin (FlexX scores: reduced gliotoxin: –2.56 (WT) versus –2.43 kcal/mol (MT), C3-mono-thiomethylated gliotoxin: –1.15 vs –0.77 kcal/mol; MM-PBSA/MM-GBSA: reduced gliotoxin: –27.54/–36.32 (WT) versus –22.44/–27.13 kcal/mol (MT), C3-mono-thiomethylated gliotoxin: –24.20/– 31.06 vs –21.57/–27.65 kcal/mol). S-MTs like TmtA are rare in nature, especially in non26 ribosomal peptide scaffolds . Some S-MTs act independently of SAM and rely on a catalytic zinc ion, for example betaine27, 28 homocysteine S-MT and methionine synthase. Among confirmed SAM-dependent S-MTs, structural data are availa25 ble for thiopurine S-MTs and a bacterial S-MT engaged in 29 echinomycin biosynthesis. Both require close proximity of the reactants to catalyse alkylation. TmtA employs a similar mode of action, but according to phylogenetic analyses it

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belongs to a separate new clade of MTs. The presence of homologues in various fungi, including both ETP and nonETP producers, suggests that during evolution TmtA mayhave been hired for fine-tuning of ETP biosynthesis by pro16 ducing bis(methylthio)gliotoxin as a regulatory metabolite. Moreover, TmtA relatives may account for S-alkylation of various ETPs (Figure 1b) as well as of bacterial toxins. Thi30 oether formation has also been described for holomycin, a member of the dithiolopyrrolone class of bacterial antibiotics, but the S-MT in charge has not been identified so far. Smethylation of secondary metabolites thus may serve as a universal self-protection system of toxin producers by depleting harmful bioactivity of the compounds and/or by repressing expression of their biosynthetic gene clusters. In summary, we determined the crystal structure of the first bis-thiomethyltransferase, TmtA, a fungal S-MT involved in the modification of gliotoxin. The elucidated structural features of TmtA classify this enzyme as a SAMdependant class-I small molecule MT. Molecular docking studies provide insights into substrate binding, stepwise conversion as well as product release. Despite its low affinity, gliotoxin undergoes specific interactions with the active site cavity and most likely serves as the primary substrate for TmtA. Its conformation in the substrate binding pocket implies that the first methyl group is transferred to the C10a sulfur atom. In a second reaction, the C3-SH group is alkylated to yield bis(methylthio)gliotoxin (Figure 4). This methylation sequence is supported by computationally obtained protein:ligand complex structures and free energy calculations of binding for reduced gliotoxin as well as its monoand bis-thiomethylated derivatives. Understanding the enzymatic production of bis-methylthiogliotoxin may also be valuable for its potential use as a clinical marker for invasive aspergillosis. Since TmtA is encoded outside the gli gene cluster, it may have evolved later for back-up self-protection and fine tuning of gliotoxin production. Identification of



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TmtA-homologues from various microbes including non-ETP producers and their biochemical characterization may shed further light on this exceptional type of S-MT, its substrate specificity and on the function of S-methylation as a detoxification and regulatory strategy. METHODS Experimental procedures for computational studies are described in detail in the Supporting Information. Mutagenesis. TmtA gene variants were created by QuikChange mutagenesis using the pET43.1 H6-TEV-TmtA plas15 mid , provided by Dr. D. Scharf (Hans Knöll Institute, Jena, Germany), as a template (Primer sequences are given in Table S5). Introduction of the respective mutations was confirmed by Sanger sequencing (GATC Biotech). Expression of WT and mutant TmtA. The plasmids encoding WT or mutant TmtA were transformed into E. coli BL21 (DE3). Bacterial cultures were grown in LB medium -1 supplemented with 180 mg L ampicillin (Amp) at 37 °C to an optical density (OD600) of 0.7. The cultures were cooled to 25 °C and isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added in a final concentration of 0.5 mM to induce gene expression. After 4 h of incubation at 25 °C the cells were harvested by centrifugation, washed with 0.9% (w/v) NaCl, and frozen at -20 °C. Selenomethionine labeling. Selenomethionine labeled protein was expressed in E. coli BL21 (DE3) at 25 °C according 31 to the protocol of Van Duyne et al. A preculture in LBAmp medium was used to inculate a second preculture 1:100 in M9 -1 medium containing 180 mg L ampicillin. Both were incubated at 37 °C. 3 L of minimal medium M9, supplemented with 0.4% (w/v) glucose, 2 mM MgSO4, vitamins, trace elements -1 and 180 mg L ampicillin were inoculated 1:50. Cells were grown to an OD600 of 0.6 at 37 °C. The culture was cooled down to the expression temperature of 25 °C and a mixture -1 -1 of 0.05 g L SeMet (Calbiochem, San Diego, CA), 0.1 g L -1 lysine, threonine, and phenylalanine as well as 0.5 g L leucine, isoleucine, and valine was added. 15 min later protein expression was induced with 0.5 mM IPTG. After 4 h of incubation at 25 °C the cells were harvested by centrifugation, washed with 0.9% (w/v) NaCl, and frozen at –20 °C. Purification of WT and mutant TmtA. Frozen bacterial cells were thawed, resuspended in 100 mM Tris/HCl pH 7.5, 500 mM NaCl, 20 mM imidazole (buffer A) and lysed by sonification. The resulting suspension was centrifuged at 21,000 rpm for 30 min at 4 °C. The clear supernatant was loaded on a 5 mL nickel chelating Sepharose FF column (GE Healthcare Life Science), which had been equilibrated with buffer A (flow rate 5 mL/min). Unbound or loosely associated proteins were removed by extensive washing with buffer A. The protein was eluted by applying a 50 ml linear gradient from buffer A to buffer B (100 mM Tris/HCl pH 7.5, 500 mM NaCl, 500 mM imidazole). Protein containing fractions were identified by SDS-PAGE, pooled and dialyzed against buffer C (20 mM Tris/HCl pH 7.5, 100 mM NaCl) for 2 h at 4 °C. Next, tobacco etch virus (TEV) protease was added in a molar stoichiometry of 1:20 to cleave the N-terminal His6-tag and the mixture was dialyzed overnight at 4 °C. To remove residual contaminations, the sample was loaded on a Superdex75 HiLoad 16/60 prep-grade size exclusion column using buffer C. TmtA eluted as a single peak. SDS-PAGE confirmed the purity of the protein (Supporting Figure 2d).

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Analysis of oligomerization state. The retention volume of TmtA on a Superdex75 10/300 GL (24 ml) size exclusion chromatography column was compared to standard proteins (LMW Gel Filtration Calibration Kit, GE Healthcare and Protein molecular weight standards, Serva) using buffer C. For four standard proteins (aprotinin, ribonuclease, carboanhydrase and bovine albumin) the log (molecular weight) values were plotted against the retention volumes and fitted to a straight line. This calibration curve and the elution volume of TmtA was used to calculate the molecular mass of the S-methyltransferase. Effects of gliotoxin on the oligomerization state of TmtA were examined by gel filtration analysis in the presence of bis-thiomethylated gliotoxin. Purified TmtA was mixed in a 2:1 molar ratio with bis(methylthio)gliotoxin (Santa Cruz Biotechnology) and incubated for 2.5 hours at 4 °C prior to gel filtration. Thermofluor based thermal shift assay. 1 μL of the fluorescent dye indicator SYPRO Orange (Sigma Aldrich, 5000 x in DMSO, 1:50 diluted in H2O) and 5 μg of purified protein -1 (13 mg mL ) were added per well in a 96-well thin-wall PCR plate (nerbe plus). Various buffers containing 100 mM buffer, pH 6–9, 50–300 mM NaCl, 0–10% (v/v) glycerol and 0–5 mM MgCl2 were used to adjust the total volume per well to 20 µL. The plate was sealed and equilibrated to 25 °C for 10 min. Afterwards the samples were heated in a Stratagene Mx 3000P qPCR System (Agilent) from 25 to 95 °C in increments of 0.5 °C/min and the fluorescence was monitored using a CCD camera (Ex 492 nm/Em 610 nm). The melting point TM for each sample was determined by fitting the data (Boltzmann sigmoidal fit (least squares)) with GraphPad Prism 5. Data points before the minimum and after the maximum of 32 fluorescence intensity were excluded from fitting. 15 Reduction of gliotoxin. Following a known procedure, NaBH4 (9 mg, 0.24 mmol) was added to a suspension of oxidized gliotoxin (10 mg, 0.031 mmol) in 2-propanol (0.75 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 45 min until full consumption of the starting material was observed. 1 M HCl was added dropwise, and the mixture was diluted with EtOAc. The aqueous layer was extracted three times with EtOAc, the combined organic fractions were dried over Na2SO4 and concentrated under reduced pressure to give reduced gliotoxin (8.2 mg, 81%). Reduction of gliotoxin was confirmed by low resolution HPLC-MS (ESI): RT = 1.93 + + min, m/z calcd for C13H17N2O4S2 [(M+H) ]: 329.06, found: 328.80. (Starting material: RT = 2.16 min, m/z calcd for + + C13H15N2O4S2 [(M+H) ]: 327.05, found: 326.72). Note: Since, we observed rapid decomposition of reduced gliotoxin in DMSO, we conducted activity assays immediately after reduction without freezing and storing the compound. Low-resolution HPLC-MS. Reduced gliotoxin was analyzed on a Dionex UltiMate 3000 HPLC system coupled to a ThermoScientific LCQ Fleet mass spectrometer with an ESI source. Eluent A consisted of water with 0.1% formic acid, eluent B consisted of 100% acetonitrile with 0.1% formic acid. The sample was injected on a C18 column (Accucore AQ, 50 x -1 2.1 mm; flow rate 0.9 ml min ) with a gradient from 5% to 95% B over 5 min. Methyltransferase activity assay. Methyltransferase activity was monitored at 37 °C with the SAMfluoro Methyltransferase Assay (G-Bioscience) according to the manufacturer’s instructions (λex of 535 nm, λem 595 nm, Tecan Spark™ 10M Multimode Microplate Reader). The final concentrations


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of protein and SAM were kept constant at 1 µM and 166 µM, respectively. The concentration of reduced gliotoxin (dissolved in DMSO) was 600 µM. The final percentage of DMSO in all samples was 2.6% (v/v). Note that enzymatic activity for the WT protein was detectable only when freshly reduced gliotoxin was used. Crystallization and structure determination. TmtA -1 was concentrated to 13 mg mL using Vivaspin centrifugal concentrators (cutoff 10 kDa). After the addition of SAM or SAH in final concentrations of 5 mM and 1 mM, respectively, the samples were stored for 1 h at 4 °C before setting up crystallization trials. Drops contained either a 1:1, 2:1 or 1:2 ratio of protein and reservoir solution. TmtA crystals grew within few days by the sitting drop vapor diffusion method from several reservoir solutions containing ammonium sulfate. Native crystals preferentially grew in 0.2 M potassium acetate and 2.2 M ammonium sulfate, whereas the best selenomethionine labeled crystals were obtained with 0.2 M cesium chloride and 2.2 M ammonium sulfate. Crystals were cryoprotected by a 1:1 mixture of mother liquor and 60% (v/v) glycerol and subsequently super-cooled in a stream of nitrogen gas at 100 K. Native and anomalous diffraction data were collected at the beamline X06SA, Swiss Light Source, Villigen, Switzerland at λ=1.0 Å and the peak wavelength of Se (λ = 0.9791 Å), respectively. Reflection intensities were evaluat33 ed with the program package XDS. 14 selenium sites were 34 35 located with SHELXD. Phasing with SHARP and solvent 36 flattening with SOLOMON resulted in an interpretable, continuous electron density map, which was automatically 37 traced with ARP/wARP and manually completed with 38 Coot. Restrained and TLS (Translation/Libration/Screw) 39 refinements with REFMAC5 yielded excellent Rwork and Rfree as well as r.m.s.d. bond and angle values. The TmtA model was proven to fulfill the Ramachandran plot using 40 PROCHECK (Table S1). Water molecules were modeled 41 with ARP/wARP solvent . Interface areas were calculated with PISA (‘Protein interfaces, surfaces and assemblies’ ser42 vice PISA at the European Bioinformatics Institute). Graph43 ical illustrations were created with PyMOL. ACCESSION CODE Structure factors and model coordinates for TmtA have been deposited in the RCSB Protein Data Bank under the accession code: 5EGP. ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website and contains Supporting Experimental Procedures, Supporting Figures, Supporting Tables and Supporting References AUTHOR INFORMATION Corresponding Author

*[email protected] Author Contributions ‡

E.R.D. and M.G. contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thank A. Brakhage, C. Hertweck and D. Scharf (Hans Knöll Institute, Jena, Germany) for providing the TmtA expression plasmid and oxidized gliotoxin. We are grateful to the staff of the beamline X06SA, SLS, Villigen, Switzerland for assistance during data collection. Financial support by the Hans-Fischer-Gesellschaft (E.M.H.) and the SFB1035 (I.A., M.G.) is acknowledged.

REFERENCES (1) Dagenais, T. R., and Keller, N. P. (2009) Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis, Clin. Microbiol. Rev. 22, 447–465. (2) Reeves, E. P., Messina, C. G., Doyle, S., and Kavanagh, K. (2004) Correlation between gliotoxin production and virulence of Aspergillus fumigatus in Galleria mellonella, Mycopathologia 158, 73–79. (3) Sugui, J. A., Pardo, J., Chang, Y. C., Zarember, K. A., Nardone, G., Galvez, E. M., Mullbacher, A., Gallin, J. I., Simon, M. M., and KwonChung, K. J. (2007) Gliotoxin is a virulence factor of Aspergillus fumigatus: gliP deletion attenuates virulence in mice immunosuppressed with hydrocortisone, Eukaryot. Cell 6, 1562–1569. (4) Kwon-Chung, K. J., and Sugui, J. A. (2009) What do we know about the role of gliotoxin in the pathobiology of Aspergillus fumigatus?, Med. Mycol. 47 Suppl. 1, S97–103. (5) Gardiner, D. M., Waring, P., and Howlett, B. J. (2005) The epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis, Microbiology 151, 1021– 1032. (6) Balibar, C. J., and Walsh, C. T. (2006) GliP, a multimodular nonribosomal peptide synthetase in Aspergillus fumigatus, makes the diketopiperazine scaffold of gliotoxin, Biochemistry 45, 15029–15038. (7) Scharf, D. H., Remme, N., Habel, A., Chankhamjon, P., Scherlach, K., Heinekamp, T., Hortschansky, P., Brakhage, A. A., and Hertweck, C. (2011) A dedicated glutathione S-transferase mediates carbon-sulfur bond formation in gliotoxin biosynthesis, J. Am. Chem. Soc. 133, 12322–12325. (8) Scharf, D. H., Chankhamjon, P., Scherlach, K., Heinekamp, T., Roth, M., Brakhage, A. A., and Hertweck, C. (2012) Epidithiol formation by an unprecedented twin carbon-sulfur lyase in the gliotoxin pathway, Angew. Chem. Int. Ed. Engl. 51, 10064–10068. (9) Scharf, D. H., Groll, M., Habel, A., Heinekamp, T., Hertweck, C., Brakhage, A. A., and Huber, E. M. (2014) Flavoenzyme-catalyzed formation of disulfide bonds in natural products, Angew. Chem. Int. Ed. Engl. 53, 2221-2224. (10) Scharf, D. H., Remme, N., Heinekamp, T., Hortschansky, P., Brakhage, A. A., and Hertweck, C. (2010) Transannular disulfide formation in gliotoxin biosynthesis and its role in self-resistance of the human pathogen Aspergillus fumigatus, J. Am. Chem. Soc. 132, 10136– 10141. (11) Schrettl, M., Carberry, S., Kavanagh, K., Haas, H., Jones, G. W., O'Brien, J., Nolan, A., Stephens, J., Fenelon, O., and Doyle, S. (2010) Self-protection against gliotoxin--a component of the gliotoxin biosynthetic cluster, GliT, completely protects Aspergillus fumigatus against exogenous gliotoxin, PLoS Path. 6, e1000952. (12) Welch, T. R., and Williams, R. M. (2014) Epidithiodioxopiperazines. occurrence, synthesis and biogenesis, Nat. Prod. Rep. 31, 1376–1404. (13) Zheng, C. J., Kim, C. J., Bae, K. S., Kim, Y. H., and Kim, W. G. (2006) Bionectins A-C, epidithiodioxopiperazines with anti-MRSA activity, from Bionectra byssicola F120, J. Nat. Prod. 69, 1816–1819. (14) Park, H. B., Kim, Y. J., Park, J. S., Yang, H. O., Lee, K. R., and Kwon, H. C. (2011) Glionitrin B, a cancer invasion inhibitory diketopiperazine produced by microbial coculture, J. Nat. Prod. 74, 2309– 2312. (15) Scharf, D. H., Habel, A., Heinekamp, T., Brakhage, A. A., and Hertweck, C. (2014) Opposed effects of enzymatic gliotoxin N- and Smethylations, J. Am. Chem. Soc. 136, 11674–11679. (16) Dolan, S. K., Owens, R. A., O'Keeffe, G., Hammel, S., Fitzpatrick, D. A., Jones, G. W., and Doyle, S. (2014) Regulation of nonribosomal peptide synthesis: bis-thiomethylation attenuates gliotoxin biosynthesis in Aspergillus fumigatus, Chem. Biol. 21, 999–1012. (17) Sun, Y., Takada, K., Takemoto, Y., Yoshida, M., Nogi, Y., Okada, S., and Matsunaga, S. (2012) Gliotoxin analogues from a marine-derived



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

fungus, Penicillium sp., and their cytotoxic and histone methyltransferase inhibitory activities, J. Nat. Prod. 75, 111–114. (18) Li, X., Kim, S. K., Nam, K. W., Kang, J. S., Choi, H. D., and Son, B. W. (2006) A new antibacterial dioxopiperazine alkaloid related to gliotoxin from a marine isolate of the fungus Pseudallescheria, J. Antibiot. (Tokyo) 59, 248–250. (19) Domingo, M. P., Colmenarejo, C., Martinez-Lostao, L., Mullbacher, A., Jarne, C., Revillo, M. J., Delgado, P., Roc, L., Meis, J. F., Rezusta, A., Pardo, J., and Galvez, E. M. (2012) Bis(methyl)gliotoxin proves to be a more stable and reliable marker for invasive aspergillosis than gliotoxin and suitable for use in diagnosis, Diagn. Microbiol. Infect. Dis. 73, 57–64. (20) Vidal-Garcia, M., Domingo, M. P., De Rueda, B., Roc, L., Delgado, M. P., Revillo, M. J., Pardo, J., Galvez, E. M., and Rezusta, A. (2015) Clinical validity of bis(methylthio)gliotoxin for the diagnosis of invasive aspergillosis, Appl. Microbiol. Biotechnol. (21) Liscombe, D. K., Louie, G. V., and Noel, J. P. (2012) Architectures, mechanisms and molecular evolution of natural product methyltransferases, Nat. Prod. Rep. 29, 1238–1250. (22) Martin, J. L., and McMillan, F. M. (2002) SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold, Curr. Opin. Struct. Biol. 12, 783–793. (23) Fauman, E. B., Blumenthal, R. M., and Cheng, X., (Eds.) (1999) Structure and evolution of AdoMet-dependent methyltransferases. , World Scientific Publishing. (24) Dai, Y. N., Zhou, K., Cao, D. D., Jiang, Y. L., Meng, F., Chi, C. B., Ren, Y. M., Chen, Y., and Zhou, C. Z. (2014) Crystal structures and catalytic mechanism of the C-methyltransferase Coq5 provide insights into a key step of the yeast coenzyme Q synthesis pathway, Acta Crystallogr. Sect. D - Biol. Crystallogr. 70, 2085–2092. (25) Peng, Y., Feng, Q., Wilk, D., Adjei, A. A., Salavaggione, O. E., Weinshilboum, R. M., and Yee, V. C. (2008) Structural basis of substrate recognition in thiopurine s-methyltransferase, Biochemistry 47, 6216– 6225. (26) Al-Mestarihi, A. H., Villamizar, G., Fernandez, J., Zolova, O. E., Lombo, F., and Garneau-Tsodikova, S. (2014) Adenylation and Smethylation of cysteine by the bifunctional enzyme TioN in thiocoraline biosynthesis, J. Am. Chem. Soc. 136, 17350–17354. (27) Evans, J. C., Huddler, D. P., Jiracek, J., Castro, C., Millian, N. S., Garrow, T. A., and Ludwig, M. L. (2002) Betaine-homocysteine methyltransferase: zinc in a distorted barrel, Structure 10, 1159–1171. (28) Matthews, R. G., and Goulding, C. W. (1997) Enzyme-catalyzed methyl transfers to thiols: the role of zinc, Curr. Opin. Chem. Biol. 1, 332– 339. (29) Hotta, K., Keegan, R. M., Ranganathan, S., Fang, M., Bibby, J., Winn, M. D., Sato, M., Lian, M., Watanabe, K., Rigden, D. J., and Kim, C. Y. (2014) Conversion of a disulfide bond into a thioacetal group during echinomycin biosynthesis, Angew. Chem. Int. Ed. Engl. 53, 824–828. (30) Li, B., Forseth, R. R., Bowers, A. A., Schroeder, F. C., and Walsh, C. T. (2012) A backup plan for self-protection: S-methylation of holomycin biosynthetic intermediates in Streptomyces clavuligerus, Chembiochem 13, 2521–2526. (31) Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J. (1993) Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin, J. Mol. Biol. 229, 105– 124. (32) Niesen, F. H., Berglund, H., and Vedadi, M. (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability, Nat. Protoc. 2, 2212–2221. (33) Kabsch, W. (2010) XDS, Acta Crystallogr. Sect. D - Biol. Crystallogr. 66, 125–132. (34) Schneider, T. R., and Sheldrick, G. M. (2002) Substructure solution with SHELXD, Acta Crystallogr. Sect. D - Biol. Crystallogr. 58, 1772– 1779. (35) de La Fortelle, E., and Bricogne, G. (1997) Meth. Enzymol. 276, 472– 494. (36) Cowtan, K. D., and Main, P. (1996) Phase combination and cross validation in iterated density-modification calculations, Acta Crystallogr. Sect. D - Biol. Crystallogr. 52, 43–48. (37) Langer, G., Cohen, S. X., Lamzin, V. S., and Perrakis, A. (2008) Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7, Nat. Protoc. 3, 1171–1179. (38) Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot, Acta Crystallogr. Sect. D - Biol. Crystallogr. 66, 486–501.

Page 8 of 9

(39) Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method, Acta Crystallogr. Sect. D - Biol. Crystallogr. 53, 240–255. (40) Laskowski, R. A., Moss, D. S., and Thornton, J. M. (1993) Mainchain bond lengths and bond angles in protein structures, J. Mol. Biol. 231, 1049–1067. (41) Perrakis, A., Sixma, T. K., Wilson, K. S., and Lamzin, V. S. (1997) wARP: improvement and extension of crystallographic phases by weighted averaging of multiple-refined dummy atomic models, Acta Crystallogr. Sect. D - Biol. Crystallogr. 53, 448–455. (42) Krissinel, E., Henrick, K. (2005) Detection of Protein Assemblies in Crystals, CompLife 2005 3695, 163–174. (43) DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA, USA.


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TOC 40x41mm (299 x 299 DPI)


Sequential Inactivation of Gliotoxin by the S-Methyltransferase TmtA

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