The enzyme from Saccharomyces cerevisiae specifically methylates guanine9 [1,2]. The bifunctional enzyme from Thermococcus kodakaraensis also catalyses the methylation of adenine9 in tRNA (cf. EC 2.1.1.218, tRNA (adenine9-N1)-methyltransferase) .
The taxonomic range for the selected organisms is: Thermococcus kodakarensis The expected taxonomic range for this enzyme is: Eukaryota, Archaea, Bacteria
The enzyme from Saccharomyces cerevisiae specifically methylates guanine9 [1,2]. The bifunctional enzyme from Thermococcus kodakaraensis also catalyses the methylation of adenine9 in tRNA (cf. EC 2.1.1.218, tRNA (adenine9-N1)-methyltransferase) [1].
formation of N1-methylguanine9 in tRNA(Asp) from Thermococcus kodakaraensis that contains a guanosine at position 9. The enzyme forms approximately the same amount of m1A and m1G when the tRNA of the yeast strain Y16243 is used as substrate. Given that occurrence of A9 and G9 in this tRNA population is almost equal (about 50% each) this result indicates that the enzyme TK0422p does not show any preference for one of these two nucleosides. The ratio m1A/m1G formed from Escherichia coli tRNA is higher than that with tRNA from the yeast Y16243 strain. This is consistent with the fact that there are about two times more tRNAs with A9 than with G9 in Escherichia coli. The enzyme is active in a pH range 5.5-9.75. The intensity of m1A and m1G spots varies greatly as a function of the pH. At pH 5.5, m1A MTase activity of TK0422p is predominant over m1G. At pH 7 or higher, both m1A and m1G are detected, m1G intensity growing with increasing pH
usage of [alpha-32P]-labeled tRNA substrates. The enzyme shows activity with both guanine9 and adenine9 containing tRNAs for methylation on N1. Bifunctional enzymes (catalyzing both m1A9 and m1G9) share the same rate-determining step for methylation as the monofunctional enzyme, these enzymes would also exhibit a different pattern of pH dependence for the two methylation reactions because of the difference in N1 pKa between adenine versus guanine
usage of [alpha-32P]-labeled tRNA substrates. The enzyme shows activity with both guanine9 and adenine9 containing tRNAs for methylation on N1. Bifunctional enzymes (catalyzing both m1A9 and m1G9) share the same rate-determining step for methylation as the monofunctional enzyme, these enzymes would also exhibit a different pattern of pH dependence for the two methylation reactions because of the difference in N1 pKa between adenine versus guanine
required, the two tRNAs from Saccharomyces cerevisiae requires a much higher Mg2+ concentration (6-10 mM in the assay) for maximal TkTrm10 activity compared to the two Thermococcus kodakarensis tRNAs, for which maximal activity is observed at about 1 mM Mg2+ or less. Similar trends are exhibited for both m1G9 and m1A9 reactions, indicating that the identity of the target purine does not affect the observed metal dependencies
the bifunctional enzyme is active in a pH range 5.5-9.75. The intensity of m1A and m1G spots varies greatly as a function of the pH. At pH 5.5, m1A MTase activity of TK0422p is predominant over m1G. At pH 7 or higher, both m1A and m1G are detected, m1G intensity growing with increasing pH
aside from an active site aspartate residue, alignment of the available Trm10 protein structures and their primary sequences show no other obvious amino acid candidates in the active site that could account for the differences between m1G9-specific (Saccharomyces cerevisiae and Schizosaccharomyces pombe), m1A9-specific (Sulfolobus acidocaldarius) and m1A9/m1G9 dual-specific (human Trmt10C and Trm10 from Thermococcus kodakarensis) Trm10 MTases. It is possible that the purine specificity might simply be due to differences in surface charge around the active site and size and/or layout of the purine-binding pocket, which could allow different Trm10 family members to accommodate different purine substrates, rather than to specific residues for catalysis. The active site pocket is more open for the m1G9-specific Trmt10A and m1A9-specific Trm10, compared to the other Trm10 proteins. No obvious similarities are observed within the m1G9-specific group of proteins that are also clearly different from the m1A9-specific Trm10, and altered in the m1G9/m1A9 dual-specific protein
the methylation on the N1 atom of adenosine to form 1-methyladenosine (m1A) has been identified at nucleotide position 9, 14, 22, 57, and 58 in different tRNAs. In some cases, these modifications have been shown to increase tRNA structural stability and induce correct tRNA folding. The m1A9 MTases belong to the Trm10 subfamily of the SPOUT superfamily. In addition to the m1A9 modification, the Trm10 subfamily of MTases methylates guanosine in some organisms
site-directed mutagenesis, the mutation has no significant effect on the A-preference for TktRNAAsp, but exhibits a modest, but shows about 4fold reduced G-preference activity compared to wild-type