Reagents

Potassium tellurite was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Dimethyl ditelluride, the source of methanetellurol, was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Since there was no commercially available reagent for dimethyltelluride and trimethyltelluronium ion, we synthesized these methylated Te compounds as described in the following section. S-Adenosyl methionine (SAM) as a methyl group donor was purchased from New England Biolabs Japan, Inc. (Tokyo, Japan). Reduced glutathione (Nacalai Tesque, Inc., Kyoto, Japan) was used as a reducing agent to generate telluride and/or tellurodiglutathione, a potential substrate for TPMT, following a previous study of selenium (Fukumoto et al. 2020). Hydrogen peroxide (FUJIFILM Wako Pure Chemical Corporation) was used to transform volatile Te compounds, such as methanetellurol and dimethyltelluride, into less volatile oxidized forms. Milli-Q water with a specific resistance of 18.2 MΩ·cm was used throughout the experiments (Merck Millipore, Burlington, MA, USA).

Synthesis of methylated Te compounds

Dimethyltelluride was prepared from elemental Te following the method described below. Powdered elemental Te (Strem, Newburyport, MA, USA) was suspended in four equivalents of methyl iodide (Nacalai Tesque, Inc.) and the mixture was heated at 80 °C for 48 h. Elemental Te was completely dissolved after heating, and a red precipitate was produced. The precipitate was collected by washing with chloroform (Nacalai Tesque, Inc.) to remove residual methyl iodide. For trimethylated form, we used trimethyltelluronium iodide synthesized and characterized in our previous study (Ogra et al. 2008).

Expression and purification of recombinant human TPMT and INMT

Recombinant human TPMT and INMT were expressed in Escherichia coli (E. coli). pBAD vectors with the cDNA sequence of hTPMT were transfected into TOP10 E. coli and pET29b vectors with the cDNA sequence of hINMT were transfected into BL21(DE3) E. coli by electroporation. The cells were cultured in an LB medium until the optical density at 600 nm (OD600) reached 0.5. Protein expression was induced by the addition of L-arabinose (FUJIFILM Wako Pure Chemical Corporation) for TPMT and isopropyl β-D-1-thiogalactopyranoside (IPTG, Takara Bio Inc., Shiga, Japan) for INMT. After induction for 4 h at 37 °C, the cells were collected by centrifugation. The cells were suspended in a lysis buffer (20 mM sodium phosphate, 300 mM NaCl), subjected to three freeze–thaw cycles using liquid nitrogen, and ultrasonicated 10 times at 10 s each (5 W, 28 kHz, TOMY SEIKO CO., LTD., Tokyo, Japan). To extract the proteins, 0.1% Triton X-100 was added, and the lysate was cleared by centrifugation. The histidine-tagged TPMT and INMT proteins were purified with TALON resin (Takara Bio Inc.) and finally recovered with elution buffer (20 mM sodium phosphate, 300 mM NaCl, 300 mM imidazole, 0.1% Triton X-100, pH 6.8). The collected fractions were dialyzed with a Slide-A-Lyzer Dialysis Cassette (molecular weight cutoff 10 kDa; Thermo Fisher Scientific, Waltham, MA, USA) using a dialysis buffer (20 mM Tris–HCl, 100 mM NaCl, 0.1% Triton X-100, pH 7.4) for 24 h.

Reaction of Te compounds catalyzed by TPMT and INMT

Purified TPMT and INMT were added into a test tube containing Te compounds. The reaction mixture had the following composition: 40 μg/mL TPMT and/or INMT, 1.0 μM SAM, 10 mM reduced glutathione, and 20 mM sodium phosphate buffer (pH 6.8). Te compounds potassium tellurite, dimethyl ditelluride, and dimethyltelluride were used as substrates at the concentration of 2.0 μM. The reaction was allowed to proceed at 37 °C for 24 h. This was followed by incubation with 3% hydrogen peroxide for another 30 min. As noted above, volatile Te species, such as methanetellurol and dimethyltelluride, were converted into their oxidized forms by hydrogen peroxide. Excess hydrogen peroxide was degraded with 0.1 μg/mL catalase at 37 °C for 1 h. The mixture was filtered through a 0.45 μm PTFE syringe-driven filter (Millex-LH, Merck Millipore), and the filtrate was analyzed by LC-ICP-MS.

Evaluation of methylated compounds by LC-ICP-MS

An HPLC system was used (Prominence; Shimadzu, Kyoto, Japan) in combination with an anion-exchange column (PRP-X 100, 2.1 × 250 mm, 5 μm particle size; Hamilton, Reno, NV, USA) for the separation of Te compounds. A 20 μL aliquot of the reaction mixture was applied to the column, and the column was eluted with 50 mM Tris-HCl (pH 8.2) at the flow rate of 0.4 mL/min. The eluate from HPLC was continuously introduced into an ICP-MS (Agilent 8800 ICP-MS/MS; Agilent Technologies, Tokyo, Japan). Te signal intensities was monitored at m/z 126, 128, and 130 with an integration time of 0.1 s each. The signal intensity of 128Te was used in this study. Details of instrument settings and operation conditions are summarized in Table 1.

Table 1 Instrumentation and operational settingsDetection of volatile methylated compounds by GC-MS

We confirmed the enzymatic formation of dimethylated Te compounds by gas chromatography–mass spectrometry (GC-MS) because methanetellurol and dimethyltelluride could not be separately detected by LC-ICP-MS. Following the method noted above, the reaction of potassium tellurite was performed with or without TMPT in a glass crimp vial at 37 °C for 24 h. Solid-phase microextraction (SPME) fiber (Carboxen/Polydimethylsiloxane, df 75 μm, 24 gauge; SUPELCO, Bellefonte, PA, USA) was inserted into the rubber cap to collect volatile dimethyltelluride in the headspace of the vial during the incubation. The vial was heated at 50 °C for 2 h to completely volatilize dimethyltelluride in the reaction mixture. The collected volatile sample that adsorbed to the SPME fiber was injected into GC-MS with a capillary column, DB-5 ms (30 m × 0.25 mm i.d., df 0.25 µm; Agilent J&W, Santa Clara, CA, USA). GC-MS analysis was performed using a mass selective detector coupled to a gas chromatograph (5975C GC/MSD; Agilent Technologies). Signal intensities were monitored at m/z 154, 155, 156, 158, and 160, which corresponded to the m/z of dimethyltelluride with different Te isotopes (i.e., 124Te, 125Te, 126Te, 128Te, and 130Te). Details of instrument settings and operation conditions for GC-MS are summarized in Table 1.

Assay for methyltransferase activity

To evaluate the contribution of TPMT to the methylation of tellurite and methanetellurol, methyltransferase activity was quantitatively evaluated with an MTase-Glo Methyltransferase Assay Kit (Promega, Madison, WI, USA). Purified TPMT was reacted with potassium tellurite or dimethyl ditelluride (methanetellurol) in plastic tubes in the presence of 50 μM SAM, 5 mM reduced glutathione, and 20 mM sodium phosphate buffer (pH 6.8). The concentrations of Te compounds were 0, 0.83, 8.3, and 83 μM for tellurite and 0, 1.52, 15.2, and 152 μM for methanetellurol. After the reaction for 30 min at 37 °C, 1 µL of 6 × MTase-Glo Reagent was added to 5 µL of the reaction mixture, and the mixture was incubated for 30 min at 37 °C to convert S-adenosylhomocysteine into ADP. Finally, 6 µL of MTase-Glo Detection Solution was added, and the mixture was incubated for another 30 min at 37 °C to convert ADP into ATP. The amount of ATP was determined from the luminescence intensity of luciferase using a plate reader, SpectraMax iD3 (Molecular Devices, San Jose, CA, USA).