Post-transcriptional modifications of transfer RNA (tRNA) are made to structurally diversify tRNA.  These modifications alter non-covalent interactions within the ribosomal machinery, resulting in phenotypic changes related to cell metabolism, growth, and virulence.  MiaE is a carboxylate bridged, non-heme diiron monooxygenase, which catalyzes the O2-dependent hydroxylation of a hypermodified-tRNA nucleoside at position 37 (2-methylthio-N6-isopentenyl-adenosine(37)-tRNA) [designated ms2i6A37].  Three nucleoside substrate-surrogates (i6A, Cl2i6A, and ms2i6A) and their corresponding hydroxylated products (io6A, Cl2io6A, and ms2io6A) were synthesized to investigate the chemo- and stereospecificity of this enzyme.  In the absence of the native electron transport chain, the peroxide-shunt was utilized to monitor the steady-state rate of substrate hydroxylation.  Remarkably, regardless of the substrate (i6A, Cl2i6A, and ms2i6A) used in peroxide-shunt assays, hydroxylation of the terminal isopentenyl-C4-position was observed with > 97% E-stereoselectivity. Steady-state kinetic experiments also demonstrate that the MiaE substrate-affinity for the free i6A, Cl2i6A, and ms2i6A nucleosides are largely dominated by functionality of the C2-position of the nucleoside base.  Indeed, the > 3-fold rate enhancement exhibited by MiaE for the hydroxylation of the free ms2i6A nucleoside relative to i6A is consistent with previous whole cell assays reporting the io6A and ms2io6A product distribution within native tRNA-substrates. Recent efforts have focused on the incorporation of radical spin-labels on substrate-surrogates, as well as the anticodon stem loop of tRNA, to interrogate key substrate-enzyme binding interactions relevant to native catalysis using Electron Paramagnetic Resonance (EPR).

Peroxide-shunt substrate-specificity for the <i>Salmonella typhimurium</i> O<sub>2</sub>-dependent tRNA modifying monooxygenase (MiaE)

247th National Meeting (2014)

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Peroxide-shunt substrate-specificity for the Salmonella typhimurium O2-dependent tRNA modifying monooxygenase (MiaE)

Peroxide-shunt substrate-specificity for the Salmonella typhimurium O₂-dependent tRNA modifying monooxygenase (MiaE)