Biochemical and genetic studies define the functions of methylthiotransferases in methanogenic and methanotrophic archaea

Methylthiotransferases (MTTases) are radical S-adenosylmethionine (SAM) enzymes that catalyze the addition of a methylthio (-SCH3) group to an unreactive carbon center. These enzymes are responsible for the production of 2-methylthioadenosine (ms2A) derivatives found at position A37 of select tRNAs in all domains of life. Additionally, some bacteria contain the RimO MTTase that catalyzes the methylthiolation of the S12 ribosomal protein. Although the functions of MTTases in bacteria and eukaryotes have been established via detailed genetic and biochemical studies, MTTases from the archaeal domain of life are understudied and the substrate specificity determinants of MTTases remain unclear. Here, we report the in vitro enzymatic activities of an MTTase (C4B56_06395) from a thermophilic Ca. Methanophagales anaerobic methanotroph (ANME) as well as the MTTase from a hyperthermophilic methanogen – MJ0867 from Methanocaldococcus jannaschii. Both enzymes catalyze the methylthiolation of N6-threonylcarbamoyladenosine (t6A) and N6-hydroxynorvalylcarbamoyladenosine (hn6A) residues to produce 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) and 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine (ms2hn6A), respectively. To further assess the function of archaeal MTTases, we analyzed select tRNA modifications in a model methanogen – Methanosarcina acetivorans – and generated a deletion of the MTTase-encoding gene (MA1153). We found that M. acetivorans produces ms2hn6A in exponential phase of growth, but does not produce ms2t6A in detectable amounts. Upon deletion of MA1153, the ms2A modification was absent, thus confirming the function of MtaB-family MTTases in generating ms2hn6A modified nucleosides in select tRNAs.

agar plates containing ampicillin (50 µg/mL).MJ0867 (UniProt Accession: A0A832SY65; NCBI Accession: WP_064496643) -the MTTase from M. jannaschii -was first cloned into pET15b by PCR amplification of the gene using primers designed with associated overlaps for Gibson assembly (Table 2) and with genomic DNA from M. jannaschii as a template.Since MJ0867 did not express well in our routine E. coli expression strain for methanogen proteins (E. coli-CodonPlus (DE3)-RIL), this putative MTTase was subsequently cloned into pJAR50CT with a Cterminal twin strep tag (pKB604, see methods for construction of this plasmid below) for expression in M. maripaludis (see Table 2 for primers).
The sequences of all constructs were verified by Sanger sequencing by the Genomics Sequencing Center at Virginia Tech and/or by Plasmidsaurus (Lexington, KY).
Construction of pKB604.To replace the C-terminal his-tag of pJAR50CT with a Cterminal twin strep tag, the plasmid backbone was first PCR amplified such that the his-tag was removed.The forward primer was designed to bind downstream of the his-tag and contained a 5' AscI restriction site.The reverse primer was designed to bind upstream of the his-tag and contained a 5' NdeI restriction site.This replaced the NsiI site with an NdeI site.To clone in the twin strep tag, a gBlock was designed containing a thrombin site and the strep tag, as well as regions overlapping with the plasmid backbone.The gBlock and 5,025 bp PCR product were assembled using NEBuilder HiFi DNA Assembly Master Mix followed by transformation into E. coli DH5a cells.The correct sequence of the cloning region was verified via Sanger sequencing.
High-resolution LC-MS analysis of hn 6 A in M. acetivorans.To confirm the identity of hn 6 A as opposed to m 6 t 6 A in M. acetivorans, we analyzed the tRNA nucleosides on a Waters Synapt G2-S HDMS interfaced with an Acquity I-Class UPLC system with an Acquity BEH C18 column (2.1 mm x 50 mm; particle size, 1.7 µm; maintained at 35 ºC).Solvent A was water with 0.1% formic acid, and solvent B was acetonitrile with 0.1% formic acid.The flow rate was 0.2 ml/min, and gradient elution was employed in the following manner (time [min], percent solvent B): (0.01, 1), (5,20), (7,95), and (8,95).Two microliters of sample was injected.The mass spectral data were collected in high-resolution MSe continuum mode (nonselective MS/MS acquisition mode).
Parameters were a 2.8-kV capillary voltage, a 125°C source temperature, a 350°C desolvation temperature, a 35-V sampling cone, 50-liter/h cone gas flow, a 500-liter/h desolvation gas flow, and a 6-liter/h nebulizer gas flow.The collision energies for the low-energy scans (function 1) were 4 V and 2 V in the trap region and the transfer region, respectively.Collision energies for the highenergy scans (function 2) were ramped from 25 to 45 V in the trap region and 2 V in the transfer region.Data were analyzed using MassLynx software (Waters).S2).The expected size of the PCR product for the wild-type (WWM60) is 3,710 bp and the expected size for the deletion strain is 2,215 bp.60 bp at the 5' end and 60 bp at the 3' end of the gene were left intact in the design of the deletion strain.The PCR product of the deletion strain compared to wild-type was also verified by Sanger sequencing.Additionally, as mentioned in the main manuscript, this molecule elutes about 0.8 minutes after ms 2 t 6 A. This ion is observed in all tRNA preps reported in this work.

Figure S5
. Growth curve of wild-type (WWM60) compared to DMA1153 M. acetivorans strains grown in triplicate in high salt medium with 100 mM methanol at 37 °C.It is important to note that the gene editing plasmid was not cured from the DMA1153 strain investigated in this study, but it was grown here in the absence of antibiotic.A small peak exists that may be ms 2 t 6 A (asterisk), but it is in too low abundance to confirm.Overall, this control experiment shows that the activity reported in Figure S7 above and Figure 7 of the main manuscript is a result of the added tRNA and not from background RNA bound to the purified enzyme.

Figure S1 .
Figure S1.PCR confirmation of the MA1153 deletion in M. acetivorans WWM60.Primers were designed to amplify beginning 1,094 base pairs upstream and 1,121 base pairs downstream of the MA1153 gene (TableS2).The expected size of the PCR product for the wild-type (WWM60) is 3,710 bp and the expected size for the deletion strain is 2,215 bp.60 bp at the 5' end and 60 bp at the 3' end of the gene were left intact in the design of the deletion strain.The PCR product of the deletion strain compared to wild-type was also verified by Sanger sequencing.

Figure S3 .
Figure S3.High-resolution LC-MS analysis of hn 6 A in Methanosarcina acetivorans.(A) Total ion current of HR-LC-MS analysis of nucleosides derived from M. acetivorans tRNA.(B) Extracted ion chromatogram for m/z = 427, corresponding to hn 6 A. (C) Mass spectrum of m/z 427 peak.The fragment ion at m/z 136.0615 (red asterisk), corresponding to the protonated adenine nucleobase, confirms the identity of hn 6 A as opposed to m 2 t6 A , the latter of which would have a fragment ion corresponding to a methylated adenine (m/z 150.077).It is important to note that this highresolution data was obtained on a different instrument with a different column and LC program compared to other data presented in the main manuscript, so the retention times differ.

Figure S4 .
Figure S4.(A)Mass spectra of ms 2 t 6 A produced in the G60 ANME-1 MTTase in vitro enzyme reaction compared to (B) the 459 ion observed in digested tRNA from M. acetivorans.The latter spectrum lacks the characteristic base fragment ion at m/z 327 and, thus, is not ms 2 t 6 A. Additionally, as mentioned in the main manuscript, this molecule elutes about 0.8 minutes after ms 2 t 6 A. This ion is observed in all tRNA preps reported in this work.

Figure S6 .
Figure S6.Purification and UV-Vis analysis of MjMTTase.(A) SDS-PAGE gel showing MjMTTase (MJ0867) with a C-terminal twin strep tag expressed and purified from M. maripaludis.(B) UV-Vis spectra of purified MjMTTase showing the 420 nm absorbance shoulder due to the presence of [4Fe-4S] clusters as well as a significant 260 nm peak due to the presence of RNA bound the purified protein (as confirmed by LC-MS in Fig. S7).

Figure S7 .
Figure S7.In vitro enzymatic activity of MjMTTase with bulk tRNA from B. subtilis DmtaB.Top spectrum shows the activity using the NADPH/FMN reducing system (8) and the bottom spectrum shows the activity using the traditional strong chemical reductant, sodium dithionite.

Figure S8 .
Figure S8.MjMTTase control reaction with all reaction components except added tRNA.(A) Total ion current chromatogram with peaks for adenosine and guanosine highlighted, confirming that the purified enzyme contains bound RNA.(B) Extracted ion chromatograms demonstrating the absence of modified nucleosides of interest.A small peak exists that may be ms 2 t 6 A (asterisk), but it is in too low abundance to confirm.Overall, this control experiment shows that the activity reported in FigureS7above and Figure7of the main manuscript is a result of the added tRNA and not from background RNA bound to the purified enzyme.

Table S1 .
List of gBlocks used in this study.

Table S2 .
List of primers used in this study.

Table S3 .
Summary of putative MTTase-encoding genes in some ANME-1 vs. ANME-2 genomes.MJ0867 (the MtaB MTTase from M. jannaschii) was used as the BLASTp query.The same putative MTTase was identified in all genomes if other MTTases, such as bacterial MtaB, MiaB or RimO, were used as queries.

Table S4 .
Analysis of the potential radical SAM sulfur-insertion enzymes found in the G60 ANME-1 genome (3).Only one protein had significant homology to any of the enzymes, and this protein only had homology to the known MTTases.