Structural, biochemical and functional analyses of tRNA-monooxygenase enzyme MiaE from Pseudomonas putida provide insights into tRNA/MiaE interaction. Issue 17 (12th August 2020)

• Record Type: Journal Article
• Authors: Carpentier, Philippe; Leprêtre, Chloé; Basset, Christian; Douki, Thierry; Torelli, Stéphane; Duarte, Victor; Hamdane, Djemel; Fontecave, Marc; Atta, Mohamed
• Is Part Of: Nucleic acids research. Volume 48:Issue 17(2020)
• Page Start: 9918
• Page End: 9930
• Publication Date: 2020-08-12
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkaa667
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: MiaE (2-methylthio- N 6 -isopentenyl-adenosine37 -tRNA monooxygenase) is a unique non-heme diiron enzyme that catalyzes the O2 -dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide 2-methylthio- N 6 -isopentenyl-adenosine (ms 2 i 6 A37 ) at position 37 of selected tRNA molecules to produce 2-methylthio- N 6 –4-hydroxyisopentenyl-adenosine (ms 2 io 6 A37 ). 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 O2 site along a conserved hydrophobic tunnel leading to the diiron active center.