A NOVEL ENZYMATIC PATHWAY LEADING TO 1-METHYLINOSINE MODIFICATION IN HALOFERAX-VOLCANII TRANSFER-RNA

Transfer RNAs of the extreme halophile Haloferax volcanii contain several modified nucleosides, among them 1-methylpseudouridine (m(1) psi), pseudouridine (psi), 2'-O-methylcytosine (psi) and 1-methylinosine (m(1)I), present in positions 54, 55, 56 and 57 of the psi-loop, respectively, At the same positions in tRNAs from eubacteria and eukaryotes, ribothymidine (T-54), pseudouridine (psi-55), non-modified cytosine (C-56) and nan-modified adenosine or guanosine (A-57 or 0-57) are found in the so-called T psi-loop. Using as substrate a T-7 transcript of Haloferax volcanii tRNA(Ile) devoid of modified nucleosides, the enzymatic activities of several tRNA modification enzymes, including those for m(1) psi-54, psi-55, Cm-56 and m(1)I-57, were detected in cell extracts of H,volcanii, Here, we demonstrate that modification of A-57 into m(1)I-57 in H,volcanii tRNA(Ile) occurs via a two-step enzymatic process, The first step corresponds to the formation of m(1) A-57 catalyzed by a S-adenosylmethionine-dependent tRNA methyltransferase, followed by the deamination of the 6-amino group of the adenine moiety by a 1-methyladenosine-57 deaminase, This enzymatic pathway differs from that leading to the formation of m(1)I-37 in the anticodon loop of eukaryotic tRNA(Ala), In the latter case, inosine-37 formation preceeds the S-adenosylmethionine-dependent methylation of I-37 into m(1) I-37, Thus, enzymatic strategies for catalyzing the formation of 1-methylinosine in tRNAs differ in organisms from distinct evolutionary kingdoms.