Catalysis by the Second Class of tRNA(m1G37) Methyl Transferase Requires A Conserved Proline

The discovery that proteins of unrelated structures can catalyze the same enzymatic activity has important implications for the evolution of biological functions. Such unrelated proteins provide insights into nature’s capacity of creating different solutions to thermodynamic problems of the same biological reactions. Particularly significant is when unrelated proteins for the same reaction are found in different domains of life. Such events are known as non-orthologous gene displacement (1-3), which are frequently observed for DNA replication proteins, such as primases, helicases, ligases, and the repair DNA polymerases (2). Recent studies are beginning to uncover non-orthologous gene displacement for proteins that recognize RNA. For example, there are two distinct structural classes of lysyl-tRNA synthetases (LysRS) that catalyze aminoacylation of tRNA with lysine (4): the class I LysRS enzymes are prevalent in archaea, whereas class II enzymes are in bacteria and eukarya. The CCA-adding enzymes that catalyze addition of CCA to the tRNA 3′ end are also divided into two structural classes (5): class I enzymes reside in archaea, whereas class II enzymes reside in bacteria and eukarya. The widespread occurrence of such examples in protein enzymes that recognize DNA and RNA suggests that non-orthologous gene displacement is an important process in the history of decoding genetic information. We are interested in the enzyme tRNA(m1G37) methyl transferase that catalyzes transfer of a methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37 (6). The m1G37 modification is located 3′ to the anticodon in tRNAs specific for leucine (CUN codons, N being any of the four natural nucleotides), proline (CCN), one of the arginine tRNAs (CGG), and often in tRNAs that contain G37. This modification is conserved in all three domains of life for the aforementioned tRNAs, and is also present in mitochondria and chloroplasts (7). The presence of m1G37 modification has been shown to prevent frame-shifts during decoding of genetic information (7-9). Earlier genetic studies had led to the identification of the enzyme tRNA(m1G37) methyl transferase as encoded by the trmD gene in bacteria (6), and more recently the trm5 gene in yeast (10). Deletion or inactivation of trmD or trm5 has led to severe growth defect in certain bacteria and death in others (10, 11). Interestingly, the archaeal counterpart of trmD or trm5 remained elusive, until a genetic screen identified a gene of Methanocaldococcus vannielii that rescued a temperature-sensitive trmD mutant of bacteria (10). The homolog of the M. vannielii gene in M. jannaschii is Mj0883, which we have now shown as the gene that encodes the enzyme tRNA(m1G37) methyl transferase (12). Interestingly, extensive sequence and bioinformatic analysis had previously suggested that the gene product of Mj0883 would be related to those of the eukaryotic trm5 but would be distinct from those of trmD genes, such that the two proteins could be considered as descendants of unrelated families (13, 14). Our recent biochemical studies support this division, and suggest that Mj0883 and trm5 genes can be grouped into one class while trmD genes into another (12). Because of this classification, we have now renamed Mj0883 as the trm5 gene of M. jannaschii. The separation of trmD and trm5 into two classes is based on the following criteria. First, members of the proteins TrmD and Trm5, while conserved within their own class, do not share sequence similarity between the two classes (12-14). There have been three X-ray crystal structures of TrmD in recent years (15-17). These structures reveals that the TrmD enzyme is a homo-dimer, and that the highly conserved catalytic core is built by the dimer interface that binds the methyl donor AdoMet. In contrast, biochemical studies suggest that M. jannaschii Trm5 is a monomer, and that conserved residues that are likely to bind AdoMet are distinct from those of TrmD (12). The monomer structure has also been reported for the human Trm5 (18). Second, the AdoMet substrate in each subunit of the TrmD crystal structure is recognized by a trefoil knot structure (15-17), where the polypeptide chain threads through a deeply twisted loop and tucks many residues (> 15) through the loop. In the available protein data base of structural motifs that bind AdoMet, the trefoil knot structure is of the class IV fold (19), which is rare and is only observed in the SPOUT (SpoU-TrmD) enzyme family (14, 20). However, although no crystal structures exists for Trm5 at present, modeling and bioinformatics suggests that Trm5 would recognize AdoMet using the class I fold, which is one of the most common structural folds among the co-factor-dependent methyl transferases (12-14). This structural fold lacks the knotted motif, but contains an open sandwich-like domain that is found in the ancient Rossmann-fold for nucleotide-binding (21). Sequence and structural modeling of Trm5 (13, 14), together with mutational analysis (12), has established that Trm5 is evolutionarily distinct from TrmD. To gain more insights into the relationship between the two classes of tRNA(m1G37) methyl transferase, it will be necessary to elucidate and compare the catalytic motifs of each class. In the case of TrmD, the available crystal structures allow some insights into the catalytic strategy of the enzyme, although no structural information is available regarding binding of the tRNA substrate. In the case of M. jannaschii Trm5, even less is known. Of interest are the similarities and potential differences of the archaeal Trm5 from other class I methyl transferases. A relevant example is the relationship between the RNA:m5C methyl transferase and DNA:m5C methyl transferase. Although these m5C methyl transferase enzymes have the same class I AdoMet-binding fold and catalyze the same reaction, they use different sequence motifs for catalysis (22, 23). This example illustrates the possibility of sequence permutations among the class I family of methyl transferases leading to different arrangements of catalytic motifs (24). To address the deficiency in our general knowledge of archaeal Trm5, we present below a series of experiments to search for residues in Trm5 that are important for catalysis. In our previous study, using the M. jannaschii Trm5 as an example (12), we have surveyed a few conserved motifs by mutagenesis and identified a conserved proline that is critical for the activity of the enzyme. However, many other conserved motifs were not tested, raising the question of whether any of the untested residues could be important for activity. Additionally, of residues that are important for activity (such as the aforementioned proline), we wish to determine if they contribute to catalysis or to substrate binding. In this study, we have developed new tools to evaluate substrate binding and catalytic competence of the wild-type and mutants of M. jannaschii Trm5. In particular, we have established conditions of transient-state kinetics that enables us to assess catalysis in the active site of the enzyme. Results of these studies, together with those based on steady-state kinetic analysis, represent an important step forward to understand the catalytic mechanism of an archaeal Trm5.