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

Abstract

MiaE (2-methylthio-N6-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-N6-isopentenyl-adenosine (ms2i6A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N6–4-hydroxyisopentenyl-adenosine (ms2io6A37). 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.

Biochemical and Structural Aspects of Cytokinin Biosynthesis and Degradation in Bacteria

“…Yet, trans -selective hydroxylation of tRNA-bound iPR or 2MeSiPR by the same enzyme was reported by others [ 62 , 63 ]. The origin of zeatins in the tRNA of some organisms is entirely unclear, as the presence of the miaE gene is reportedly limited to a few bacterial genera [ 64 ]; however, up-to-date results of BLASTp searches (not shown) using MiaE from Pseudomonas putida [ 65 ] as a query suggests the far wider distribution of MiaE among bacteria than previously anticipated.…”

A miniature airlock system to aid the cryo-cooling of protein crystals grown under anoxic conditions

“…S4) were collected on beamline MASSIF-3 under cryogenic conditions. Integration and refinement statistics (Table 1) show that this crystal diffracted at relatively high resolution (d = 1.59 A ˚), and thus, the refined structure (PDB entry 8afj) reveals a level of detail that can be fairly compared with the aerobic structure of MiaE previously deposited (PDB entry 6zmb; Carpentier et al, 2020). The anoxic reduced diiron centre structure in the 2F o À F c map at the 2 level is depicted in Fig.…”

“…The enzyme Pp-MiaE from P. putida is a redox monooxygenase that catalyses the post-transcriptional hydroxylation of adenosine nucleotides at position 37 of selected tRNAs (Mathevon et al, 2007). Previous crystallographic studies revealed that the structure of the enzyme in the oxidized resting state consists of a four -helix bundle housing a diiron catalytic centre typical of the non-heme diiron monooxygenase family (Carpentier et al, 2020). For this test, we used an alternative crystallization condition of Pp-MiaE, producing two crystalline forms in large quantity, long needle-shaped and diamond-shaped crystals, which are both easy to manage.…”

“…Secondly, we prepared crystals of the well characterized oxygen-dependent enzyme MiaE Pseudomonas putida (Pp-MiaE) in its reduced form under anoxic conditions in the glovebox. Production, purification and preparation of the enzyme for crystallization screening were described in previous work (Carpentier et al, 2020). We used a new crystal form of Pp-MiaE that grew under hanging-drop conditions by mixing 1 ml of protein at 25 mg ml À1 with a 1 ml solution from the reservoir containing 0.5 M CaCl 2 , 25% PEG 6 K and 0.2 M Tris buffer at pH 8.5 and 10 mM sodium dithionite.…”

“…. ) that activate and process molecular oxygen (Romero et al, 2018;Poulos, 2014), such as the diiron-centrecontaining proteins that we are investigating (Engilberge et al, 2020;Carpentier et al, 2020). In contrast, O 2 (even at trace levels) can be detrimental for anaerobic organisms (even lethal for obligate anaerobes), and this sensitivity to oxygen might be explained at the molecular level.…”