Identification of eukaryotic and prokaryotic methylthiotransferase for biosynthesis of 2-methylthio-N6-threonylcarbamoyladenosine in tRNA

Abstract

Bacterial and eukaryotic transfer RNAs have been shown to contain hypermodified adenosine, 2-methylthio-N(6)-threonylcarbamoyladenosine, at position 37 (A(37)) adjacent to the 3'-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. Using a combination of bioinformatic sequence analysis and in vivo assay coupled to HPLC/MS technique, we have identified, from distinct sequence signatures, two methylthiotransferase (MTTase) subfamilies, designated as MtaB in bacterial cells and e-MtaB in eukaryotic and archaeal cells. Both subfamilies are responsible for the transformation of N(6)-threonylcarbamoyladenosine into 2-methylthio-N(6)-threonylcarbamoyladenosine. Recently, a variant within the human CDKAL1 gene belonging to the e-MtaB subfamily was shown to predispose for type 2 diabetes. CDKAL1 is thus the first eukaryotic MTTase identified so far. Using purified preparations of Bacillus subtilis MtaB (YqeV), a CDKAL1 bacterial homolog, we demonstrate that YqeV/CDKAL1 enzymes, as the previously studied MTTases MiaB and RimO, contain two [4Fe-4S] clusters. This work lays the foundation for elucidating the function of CDKAL1.

SCHEME 1.

Amino acid sequence alignments of MiaB, RimO, and MtaB (YqeV and Cdkal-1) MTTases from T. maritima (T.m.), E. coli (E.c.), B. subtilis (B.sm.), and Homo sapiens (H.s.). The alignment was performed with ClustalW at the EBI site. Totally conserved residues are indicated by asterisks, and conserved cysteine residues are shown in boxes. The three domains UPF0004, radical AdoMet, and TRAM are shown on the right.

SCHEME 2.

Biosynthetic pathway for ms²t⁶A.

FIGURE 1.

Phylogenomic analysis of bacterial and human radical AdoMet methylthiotransferases. The cladogram shows the inferred evolutionary distances between representatives of all homologous protein families identified in a systematic search of the human genome and 474 fully sequenced bacterial genomes (see “Materials and Methods”). The MiaB and RimO families have been biochemically characterized in previous literature. This study described initial experimental characterization of representatives from the methylthiothreonylcarbamoyladenosine transferase B (MtaB) family and the eukaryotic methylthiothreonylcarbamoyladenosine transferase B (e-MtaB) family. The methYthiotransferase-like family 1 (MTL1), which is restricted to ϵ proteobacteria, has yet to be characterized experimentally. The e-MtaB family is found in archaebacteria but not eubacteria, although the other four families are found in eubacteria but not archaebacteria. The organism count indicates the number of unrelated bacterial genomes that encode a representative of each family (i.e. after correction for redundancy in genome organization). The numbers in circles at the root of each family indicate the number of times a member of one of the other families shown here is encoded simultaneously in the same genome (without redundancy correction). The numbers in a smaller font near the branch points indicate the percent of MEGA bootstrap replicates with the illustrated relative ordering of the successive branch points. Splits between the identified families confidently precede those within the families, with 100, 78, 98, and 99% confidence. Proper grouping of proteins in the MtaB family, which has the lowest 78% confidence for its first internal split, is supported by PSI-BLAST sequence profiling (data not shown) as well as the fact that proteins assigned to this family are never encoded in the same genome, even though 198 members of the other families are found encoded in the same genome as MtaB family members. The ORF data in parentheses includes the name of the protein, its amino acid sequence length, its overall pI, and the pI of its TRAM domain. The TRAM domains of bacterial MiaBs are generally basic, although those of bacterial RimOs are generally acidic. The TRAM domains of members of the other families show wider variations in pI values.

FIGURE 2.

HPLC, UV-visible detection, and mass spectra of i⁶A, t⁶A, and ms²t⁶A modified nucleosides using E. coli. The chromatograms correspond to the analysis (45–90-min region) of bulk tRNA from the following: miaB⁻ TX3346 E. coli strain (A), complemented with pT7-mtab (B) and pT7-emtab (C). The UV-visible spectra of the i⁶A (D), t⁶A (E) and ms²t⁶A (F) and the corresponding mass t6A (G) ms²t⁶A obtained after complementation with pT7-mtab (H), pT7-emtab (I). The experiments have been run in triplicate, and the areas have been found to be reproducible within a 5% margin error. Mass spectrometry detection was carried out in neutral loss mode to obtain a high specificity as described under “Materials and Methods.” The peak denoted with asterisk corresponds to the Na⁺-protonated pseudo-molecular ions for t⁶A (G) (MH⁺ = 435.5) and ms²t⁶A (H and I) (MH⁺ = 481.3). mAU, milli-arbitrary units.

FIGURE 3.

HPLC and UV-visible detection of t⁶A, ms²t⁶A, i⁶A, and ms²i⁶A modified nucleosides using B. subtilis. The chromatograms correspond to the analysis (45–90-min region) of bulk tRNA from the following: MGNA-001 B. subtilis wild-type strain (A); MGNA-C496 B. subtilis yqeV⁻ strain (B); MGNA-C496 B. subtilis yqeV⁻ strain complemented with the pDB148-yqeV plasmid (C). The UV-visible spectra of the t⁶A (D), ms²t⁶A (E), i⁶A (F), and ms²i⁶A (G) are shown. The experiments have been run in triplicate, and the areas have been found to be reproducible within a 5% margin error. mAU, milli-arbitrary units.

FIGURE 4.

Biochemical and spectroscopic characterization of MtaB protein. A, SDS-polyacrylamide (12%) gel of MtaB after Superdex 75 chromatography (2 and 6 μg, lanes 2 and 3, respectively). Molecular weight markers are in lanes 1 and 4. B, light absorption spectra of apo-MtaB (4 μm) (trace 1) and holo-MtaB (8 μm) (trace 2) MtaB in 50 mm Tris-Cl, pH 8.0, with 50 mm KCl. C, X-band EPR spectrum of the reduced holo-MtaB (100 μm) MtaB in 50 mm Tris-Cl, pH 8.0, with 50 mm KCl. Experimental conditions are as follows: microwave power, 100 microwatts; microwave frequency, 9.6 GHz; modulation amplitude 1 millitesla; temperature 12 K.