Fig. 2: N341 is a modified cytidine conjugated with 5-AVA.
From: A tRNA modification with aminovaleramide facilitates AUA decoding in protein synthesis
a, Involvement of TilS in N341 biogenesis in V. cholerae. LC–MS analysis of total nucleosides from the V. cholerae strain (TilS expression is controlled by the arabinose promotor) cultured in the presence (top) or absence (bottom) of arabinose. MRM chromatograms detecting N341 (m/z 342 > 210), L (m/z 372 > 240) and queuosine (m/z 410 > 295) are shown. b, Metabolic labeling of N341 with stable isotope-labeled lysine. LC–MS analyses of total nucleosides from V. cholerae cells cultured in M9 medium (left) and M9 medium supplemented with full-labeled Lys (13C6,15N2-lysine; middle) and one carbon-labeled Lys (1-13C-lysine; right). XICs detecting proton adducts of N341 nucleoside with different isotopes as indicated on the right are shown. Asterisks represent labeled carbon and nitrogen atoms of Lys. c, Chemical structure (imine isomer) of ava2C. d, CID spectrum of the spinach N341 base moiety (BH2+) with parent m/z 210. The daughter ions are assigned on the ava2C base. e, Mass (left) and CID (right) spectra of N341 nucleoside in D2O solution. Eight and six exchangeable protons, including a proton for ionization, are assigned on the ava2C nucleoside (left) and its base moiety (right), respectively. The asterisk indicates a non-specific signal not derived from the D-labels. f, Schematic representation of the chemical synthesis of the ava2C nucleoside (see Methods for details). g, LC–MS co-injection analyses with chemically synthesized ava2C. Spinach N341 (top), chemically synthesized ava2C (middle) and a co-injected sample (bottom) were analyzed by an ODS column. h, LC–MS co-injection analyses with enzymatically synthesized ava2C. Spinach N341 (top), enzymatically synthesized ava2C (TilS + 5-AVA; middle) and a co-injected sample (bottom) were analyzed by an ODS column. i, Proposed biosynthetic pathway of ava2C. First, C34 is converted to L34 catalyzed by TilS using Lys and ATP as substrates. Second, L34 is further converted to ava2C by an unidentified enzyme(s).
