Structural, biochemical and functional analyses of tRNA-monooxygenase enzyme MiaE from Pseudomonas putida provide insights into tRNA/MiaE interaction

MiaE (2-methylthio-N(6)-isopentenyl-adenosine(37)-tRNA monooxygenase) is a unique non-heme diiron enzyme that catalyzes the O(2)-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide 2-methylthio-N(6)-isopentenyl-adenosine (ms(2)i(6)A(37)) at position 37 of selected tRNA molecules to produce 2-methylthio-N(6)–4-hydroxyisopentenyl-adenosine (ms(2)io(6)A(37)). Here, we report the in vivo activity, biochemical, spectroscopic characterization and X-ray crystal structure of MiaE from Pseudomonas putida. The investigation demonstrates that the putative pp-2188 gene encodes a MiaE enzyme. The structure shows that Pp-MiaE consists of a catalytic diiron(III) domain with a four alpha-helix bundle fold. A docking model of Pp-MiaE in complex with tRNA, combined with site directed mutagenesis and in vivo activity shed light on the importance of an additional linker region for substrate tRNA recognition. Finally, krypton-pressurized Pp-MiaE experiments, revealed the presence of defined O(2) site along a conserved hydrophobic tunnel leading to the diiron active center.