Steady-state kinetics and spectroscopic characterization of enzyme-tRNA interactions for the non-heme diiron tRNA-monooxygenase, MiaE.

MiaE [2-methylthio-N(6)-isopentenyl-adenosine(37)-tRNA monooxygenase] isolated from Salmonella typhimurium is a unique non-heme diiron enzyme that catalyzes the O2-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide (ms(2)i(6)A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N(6)-(4-hydroxyisopentenyl)-adenosine(37). In this work, isopentenylated tRNA substrates for MiaE were produced from small RNA oligomers corresponding to the anticodon stem loop (ACSL) region of tRNA(Trp) using recombinant MiaA and dimethylallyl pyrophosphate. Steady-state rates for MiaE-catalyzed substrate hydroxylation were determined using recombinant ferredoxin (Fd) and ferredoxin reductase (FdR) to provide a catalytic electron transport chain (ETC) using NADPH as the sole electron source. As with previously reported peroxide-shunt assays, steady-state product formation retains nearly stoichiometric (>98%) E stereoselectivity. MiaE-catalyzed i(6)A-ACSL(Trp) hydroxylation follows Michaelis-Menten saturation kinetics with kcat, KM, and V/K determined to be 0.10 ± 0.01 s(-1), 9.1 ± 1.5 µM, and ∼11000 M(-1) s(-1), respectively. While vastly slower, MiaE-catalyzed hydroxylation of free i(6)A nucleoside could also be observed using the (Fd/FdR)-ETC assay. By comparison to the V/K determined for i(6)A-ACSL substrates, an ∼6000-fold increase in enzymatic efficiency is imparted by ACSL(Trp)-MiaE interactions. The impact of substrate tRNA-MiaE interactions on protein secondary structure and active site electronic configuration was investigated using circular dichroism, dual-mode X-band electron paramagnetic resonance, and Mössbauer spectroscopies. These studies demonstrate that binding of tRNA to MiaE induces a protein conformational change that influences the electronic structure of the diiron site analogous to what has been observed for various bacterial multicomponent diiron monooxygenases upon titration with their corresponding effector proteins. These observations suggest that substrate-enzyme interactions may play a pivotal role in modulating the reactivity of the MiaE diiron active site. Moreover, the simplified monomeric (α) protein configuration exhibited by MiaE provide an unparalleled opportunity to study the impact of protein-effector interactions on non-heme diiron site geometry and reactivity.

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

Post-transcriptional modifications of tRNA are made to structurally diversify tRNA. These modifications alter noncovalent interactions within the ribosomal machinery, resulting in phenotypic changes related to cell metabolism, growth, and virulence. MiaE is a carboxylate bridged, nonheme diiron monooxygenase, which catalyzes the O2-dependent hydroxylation of a hypermodified-tRNA nucleoside at position 37 (2-methylthio-N(6)-isopentenyl-adenosine(37)-tRNA) [designated ms(2)i(6)A37]. In this work, recombinant MiaE was cloned from Salmonella typhimurium , purified to homogeneity, and characterized by UV-visible and dual-mode X-band EPR spectroscopy for comparison to other nonheme diiron enzymes. Additionally, three nucleoside substrate-surrogates (i(6)A, Cl(2)i(6)A, and ms(2)i(6)A) and their corresponding hydroxylated products (io(6)A, Cl(2)io(6)A, and ms(2)io(6)A) 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 rate of substrate hydroxylation. Remarkably, regardless of the substrate (i(6)A, Cl(2)i(6)A, and ms(2)i(6)A) used in peroxide-shunt assays, hydroxylation of the terminal isopentenyl-C4-position was observed with >97% E-stereoselectivity. No other nonspecific hydroxylation products were observed in enzymatic assays. Steady-state kinetic experiments also demonstrate that the initial rate of MiaE hydroxylation is highly influenced by the substituent at the C2-position of the nucleoside base (v0/[E] for ms(2)i(6)A > i(6)A > Cl(2)i(6)A). Indeed, the >3-fold rate enhancement exhibited by MiaE for the hydroxylation of the free ms(2)i(6)A nucleoside relative to i(6)A is consistent with previous whole cell assays reporting the ms(2)io(6)A and io(6)A product distribution within native tRNA-substrates. This observation suggests that the nucleoside C2-substituent is a key point of interaction regulating MiaE substrate specificity.