A Cobalamin‐Dependent Radical SAM Enzyme Catalyzes the Unique Cα‐Methylation of Glutamine in Methyl‐Coenzyme M Reductase

Abstract Methyl‐coenzyme M reductase, which is responsible for the production of the greenhouse gas methane during biological methane formation, carries several unique posttranslational amino acid modifications, including a 2‐(S)‐methylglutamine. The enzyme responsible for the Cα‐methylation of this glutamine is not known. Herein, we identify and characterize a cobalamin‐dependent radical SAM enzyme as the glutamine C‐methyltransferase. The recombinant protein from Methanoculleus thermophilus binds cobalamin in a base‐off, His‐off conformation and contains a single [4Fe‐4S] cluster. The cobalamin cofactor cycles between the methyl‐cob(III)alamin, cob(II)alamin and cob(I)alamin states during catalysis and produces methylated substrate, 5′‐deoxyadenosine and S‐adenosyl‐l‐homocysteine in a 1 : 1 : 1 ratio. The newly identified glutamine C‐methyltransferase belongs to the class B radical SAM methyltransferases known to catalyze challenging methylation reactions of sp3‐hybridized carbon atoms.

Results and Discussion page 6 Figure S1 page 6 Figure S2 page 7 Figure S3 page 8 Figure S4 page 9 Figure S5 page 10 Figure S6 page 11 Figure S7 page 12 Figure S8 page 13 Figure S9 page 14 Figure S10 page 14 Figure S11 page 15 24 TM bead beating lysis system (MP Biomedical) by shaking three times for 40 s at 5.5 m/s. In between, the cell suspension was cooled on ice for about 5 min. For sedimentation of the glass beads, the crude extract was centrifuged at 2790 g for 5 min at 4 °C. Afterwards, the soluble protein fraction was obtained by centrifugation at 125,082 g for 1 h at 10 °C. His6-tagged QCMT was purified by immobilized metal affinity chromatography (IMAC) using an Äkta purifier 10 FPLC system (GE Healthcare). The soluble protein fraction was loaded on a 4 ml His60 Ni Superflow Resin (Takara Bio Inc.) column equilibrated with lysis buffer B at a flow rate of 1 ml/min. Unbound proteins were washed off the column with buffer B at a flow rate of 1 ml/min until the UV signal reached a stable base line. Proteins bound to the column were eluted with 50 % elution buffer C (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 % (v/v) glycerol, 500 mM imidazole). Elution fractions were analyzed by 10 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the QCMT-containing fractions were pooled. Buffer exchange against storage buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 % glycerol) was done by using a PD-10 column (Cytiva Life Sciences) according to the manufacturer's instructions. Protein solutions were concentrated using Amicon centrifugal filter devices (Millipore) with 30 kDa cutoff.

Determination of protein concentration
Protein concentration was determined with Bradford reagent (Bio-Rad Laboratories, Inc.) using BSA as a standard according to the manufacturer's protocol.

Chemical reconstitution of cofactors
For reconstitution of the cobalamin cofactor, as-isolated QCMT (after IMAC) was incubated with equimolar amounts of either methylcobalamin or hydroxocobalamin for 1 to 2 h at room temperature. Unbound cobalamin was separated from the protein by using a PD-10 column.
Reconstitution of the iron-sulfur cluster was performed before and after size exclusion chromatography according to Flühe et al. [2] with the following modifications. Storage buffer A was used for the preparation of the required solutions. All incubation steps were done at room temperature. The protein solution was incubated with 4 -5 equiv. of ammonium iron citrate for 15 -20 min and with 4 -5 equiv. of lithium sulfide for 1 -1.5 h. After reconstitution, the protein solution was centrifuged in order to remove any precipitated material, and excess of ammonium iron citrate and lithium sulfide was removed using a PD-10 column or by size exclusion chromatography.

Size exclusion chromatography
For further purification, the reconstituted QCMT was centrifuged for 10 min (16,100 g) and loaded on a HiLoad 16/600 Superdex 200 prep grade column (GE Healthcare) equilibrated with storage buffer A at a flow rate of 1 ml/min. Elution fractions were analyzed by SDS-PAGE and the fractions containing monomeric QCMT were pooled. Analytic size exclusion chromatography was performed on Superdex 200 Increase 5/150 GL (Cytiva) equilibrated with storage buffer A at a flow rate of 0.25 ml/min. A gel filtration standard (Biorad) was employed to generate a standard curve.

Determination of iron and sulfide contents
The iron and sulfide contents of purified and reconstituted QCMT was determined as previously described. [3] UV/Visible absorption spectroscopy UV/Vis absorption spectra were recorded with a V-650 spectrophotometer (Jasco) in a quartz glass cuvette placed in an anaerobic chamber with a sample volume of 100 µl sample. Spectra were normalized by setting the absorption at 750 nm to zero. The cobalamin content of as-isolated QCMT and as-purified QCMT was analyzed by mixing 50 µM QCMT with 150 µM H2SO4 and boiling for 10 min at 70 °C. The denatured enzyme was separated by centrifugation (16,100 g, 10 min) and the supernatant was analyzed by UV/Vis spectroscopy. UV/Vis absorption spectra of titanium(III) citrate reduced QCMT were recorded after incubation of 20 µM as-purified QCMT with 80 µM titanium(III) citrate for 3 h. Then, 40 µM SAM were added to reduced QCMT and spectra were measured after incubation for an additional 30 min. Next, 20 µM peptide substrate (QS-28) were added and spectra were measured instantly. After each step, samples were taken and quenched with formic acid for HPLC analysis of SAH and DOA formation. Control reactions contained reduced QCMT with peptide substrate but without SAM, and reduced QCMT incubated with SAM and a peptide substrate with a different amino acid sequence (HAALVSMGEMLPARRARGPNEPGG). In addition, after the final step, samples were taken for LC-MS analysis of the peptide substrate. For the reduction of QCMT with sodium dithionite (DT), as-purified QCMT (9 µM) was incubated with equimolar amounts of DT for 1.5 h and spectra were measured. Then, 18 µM SAM were added and spectra were measured after incubation for 15 min.

In vitro enzyme activity assays
For the design of the peptide substrate the available amino acid sequence fragments of Methanoculleus thermophilus McrA subunit from Uniprot (https://www.uniprot.org/) were compared by multiple sequence alignments (https://www.ebi.ac.uk/Tools/msa/clustalo/). Hence, two peptides were designated as potential substrates: the peptide QS-28 consisting of the 28 amino acids QFPTMMEDHFGGSQRAGVIAAASGLSTS and the peptide PS-24 comprising the 24 amino acids PTMMEDHFGGSQRAGVIAAASGLS. These substrates were obtained by custom-synthesis (TAG Copenhagen A/S). In vitro enzyme activity assays were performed under anaerobic conditions. Reaction mixtures were set up with as-purified QCMT reconstituted with MeCbl in storage buffer A and incubated at 37 °C for 4 h unless stated otherwise. The reaction was stopped by adding formic acid [final concentration of 5 % (v/v)]. The initial in vitro reaction mixture for HPLC and LC-MS analysis contained 25 µM QCMT, 200 µM SAM, 50 µM peptide substrate (QS-28), and 1 mM sodium dithionite in a total volume of 300 µl. The final standard activity assay for HPLC analysis of SAM cleavage products contained 1 µM QCMT, 200 µM SAM, 50 µM peptide substrate (either QS-28 or PS-24), 1 mM titanium(III) citrate in a total volume of 100 µl. For analysis of the time-dependent formation of DOA and SAH a higher total volume was employed, and samples of 100 µl were taken at the respective time points and analyzed by HPLC. For quantification of SAH and DOA formation, standard solutions containing 5, 10, 15, 20, 25, 50, 75, and 100 µM of either SAH or DOA were prepared and analyzed by HPLC. For LC-MS analysis of peptides, assays were performed with 2 µM as-purified QCMT (OHCbl-Rc), 200 µM SAM, 50 µM peptide substrate (either QS-28 or PS-24), 1 mM titanium(III) citrate in a total volume of 300 µl and incubated for 3.5 h. Assays with deuteriumlabeled methionine contained 2 µM as-purified QCMT (OHCbl-Rc), 11 µM EcMAT, [4] 2 mM L-methionine (methyl-d 3 , Eurisotop), 2 mM ATP, 50 µM peptide substrate (either QS-28 or PS-24), 1 mM titanium(III) citrate, 20 mM MgCl2 and 50 mM KCl and were incubated for 4 h. Assay mixtures for determination of the pH optimum contained 1 µM QCMT, 200 µM SAM, 50 µM peptide substrate (PS-24), 1 mM titanium(III) citrate, 300 mM NaCl, 10 % (v/v) glycerol and 50 mM of one of the following buffers in a total volume of 200 µl: MES, pH 6.5; HEPES, pH 7.0; Tris-HCl, pH 7.5/ 8.0/ 8.5/ 9.0; CAPSO, pH 9.5/ 10.0; and CAPS, pH 10.5/ 11.0. Reaction mixtures were preincubated at 37 °C and started with the addition of SAM after 10 -15 min. Samples of 100 µl were quenched after 20 min and 40 min, respectively, and analyzed by HPLC. The initial velocity v0 was calculated as 0 = ∆ ∆ in µM DOA/min/µM QCMT. The assay for LC-MS analysis of enzyme-bound cobalamin contained 10 µM as-purified QCMT, 11 µM EcMAT, 1 µM EcMTAN, 2 mM L-methionine or L-d 3 -methionine, 2 mM ATP, 1 mM titanium(III) citrate, 50 µM peptide substrate, 20 mM MgCl2 and 50 mM KCl in a volume of 120 µL and were incubated for 4 h.

HPLC analysis of SAM cleavage products
Before HPLC analysis, the formic acid quenched samples were centrifuged for 10 min at 21,130 g. HPLC analysis was carried out either on a Jasco LC-2000Plus or LC-4500 series system with either a Hypercarb column (Thermo fisher, 100 x 2.1 mm, particle size 5 µm) or a Reprosil-Pur ODS-3 column (Dr. Maisch Gmbh, 125 mm x 3 mm, particle size 3 µm). The Hypercarb column was equilibrated with solvent A (double distilled water + 0.1 % (v/v) trifluoroacetic acid). Separation of the samples was done applying the following gradient at a flow rate of 0.

LC-MS and MS/MS analysis of peptide substrates
The formic acid quenched samples were centrifuged for 10 min at 21,130 g and filtered through a syringe filter (PTFE, Ø 4 mm, Pore 0.2 µm, ISERA) by centrifugation (2 -10 min, 4,500 g). For peptide LC-MS measurements, the peptides were separated from other assay components with an ISAspher 300 Å -5 µm C4 150 x 2,0 mm HPLC column on an HPLC system (Agilent 1100, USA) connected to an ABSciex Qtrap 4500 (AB Sciex, Darmstadt, Germany) hybrid ESI mass spectrometer. Mobile phase A consisted of water containing 0.1 % formic acid, and mobile phase B consisted of acetonitrile containing 0.1 % formic acid. Separation was achieved with a 40 min method using a flow rate of 0.2 ml/min. Used gradient for separation was as follows, initial: 10 % B up to 1 min, linear gradient until 8 min increasing B to 70 %, up to 15 min linear gradient increasing B to 90 %, followed by washing with 90 % B until 23 min and returning to initial conditions and re-equilibration. Mass spectrometer measurements were performed in positive ionization mode with the following parameters: Curtain gas 30 psi, ion Spray voltage 4500 V, temperature: 200 °C, entrance potential 10 V, declustering potential 100 V, nebulizer gas 40 psi, heater gas 50 psi. For measurements of peptide methylation Enhanced MS (EMS) LIT-Scan mode was performed with the following additional parameters: collisionally activated dissociation (CAD) high, collision energy 5 V and enabled dynamic fill-time. MS/MS peptide fragmentations were performed using enhanced product ion (EPI) LIT-scan mode at 27 V collision energy. For LC-MS peptide measurements 5 µl of assay mixture containing 50 µM peptide was injected. For MS/MS fragmentation after trypsinolysis, 15 µl were injected. LC-MS spectra were deconvoluted and visualized with UNiDec Version 4.2.2. [5] MS/MS spectra were visualized and edited with IPSA [6] and Inkscape (retrieved from https://inkscape.org).

Peptide substrate isolation and trypsinolysis for MS/MS analysis
The peptide was isolated from the assay mixture via HPLC (Agilent 1260 Infinity II, USA) with the same method used for LC-MS peptide measurements. The isolated peptide was freeze-dried and dissolved in 100 mM Tris-buffer pH 7.8, matching the volume injected into the HPLC before. Trypsin was added in a ratio of 1/50 (w/w) and the sample was incubated at 37 °C overnight. Desalting and separation of the digested peptide before MS/MS fragmentation was achieved by described LC method.

LC-MS analysis of amino acids from peptide hydrolysis
Before amino acid analysis via LC-MS, enzyme was removed with vivaspin 500 centrifugal concentrator (10,000 MWCO). LC-MS analysis was conducted after acid hydrolysis (6 N HCl, 120 °C, 24 h) of methylated QS-28 peptide (QCMT added) and not methylated QS-28 peptide (control lacking enzyme). Glutamine species were not detected due to the formation of glutamate under strongly acid conditions. For LC-MS measurements, the amino acids were separated with a Syngergi 4 µm Fusion-P 50 X 2 HPLC column on an HPLC system (Exion LC, Germany) connected to an ABSciex Qtrap 5500 (AB Sciex, Darmstadt, Germany) ESI mass spectrometer. Mobile phase A consisted of water containing 0.1 % formic acid, and mobile phase B consisted of acetonitrile. The duration of the method was 5 min and an isocratic of 100 % mobile Phase A was used. Mass spectrometer measurements were performed in positive ionization mode with the following parameters: Curtain gas 25 psi, ion Spray voltage 4500 V, temperature: 450 °C, entrance potential: 10 V, nebulizer gas 40 psi, heater gas 50 psi. Following MRM transitions were optimized with standards:

LC-MS analysis of enzyme-bound MeCbl
Samples were centrifuged for 15 min at 12,100 g and filtered through a syringe filter (PTFE, Ø 4 mm, Pore 0.2 µm, ISERA) for 10 min at 3,100 g. The samples were stored on ice until measurement. Measurement of enzyme-bound MeCbl was possible due to denaturing conditions during the separation on the HPLC column. Separation was achieved by running the described HPLC method from LC-MS peptide measurements. 20 µl of assay mixture containing 10 µM enzyme were injected. For mass spectrometric detection of MeCbl, precursor-Ion scan mode was performed. The Precursor was set to 665.3 Da, corresponding to the common daughter fragment of cobalamin species.