RNA biosynthesis involves many biochemical events that are essential for its maturation. From an enzymatic point of view, the nucleoside modification phenomenon is certainly one of the most remarkable of these events. It allows a multitude of structural changes in the canonical bases adenosine, guanosine, cytosine and uridine. A number of these modifications, which result from complex processes (hyper-modifications), have been characterized and are mainly located at positions 34 and 37 of transfer RNAs (tRNAs) in the so-called "anticodon" loop (Figure). They are particularly interesting because they are known to optimize the decoding function of messenger RNA by directly controlling codon-anticodon interactions. Enzymes catalyzing nucleoside modification are fundamental targets for research, as they allow the study of protein-RNA interactions and are implicated in certain diseases. Despite the wealth of genetic, physiological and biochemical data on the tRNA-modifying enzymes, very little structural data are available.

For several years, IRIG researchers have been studying the enzymes involved in the biosynthetic pathway leading to the formation of the hyper-modified nucleoside ms²io⁶A37. This hyper-modification results from a sequence of enzymatic reactions successively catalyzed by the enzymes MiaA, MiaB and MiaE, the last two of which belong to the class of metallo-enzymes. By combining biochemical, spectroscopic and crystallographic methods, IRIG researchers have identified the structural elements of MiaE that allow the docking of the enzyme with tRNA and the channeling of molecular oxygen, both of which are essential primo-processes that initiate the hydroxylation transformation.

The crystallographic structure of the Pseudomonas putida MiaE enzyme (pp-MiaE) has been obtained by the researchers. This structure is in the form of a single, compact domain that consists essentially of a bundle of four antiparallel α-helices housing a catalytic center with two iron atoms. These two metal ions are coordinated by histidines and glutamates, and bridged by an oxygen atom characterized as "μ-oxo" by spectroscopy. Two functional channels were identified. The first was mapped using krypton atoms (oxygen substitute) introduced under pressure into Pp-MiaE crystals. This channel of hydrophobic in nature, is undoubtedly the diffusion route of molecular oxygen to the active site for catalysis. The second channel, polar by nature, is interpreted as a large cavity to stereoselectively accommodate the anti-codon part of the tRNA and to position the adenine base 37 close to the active site to facilitate the hydroxylation of its isopentenyl moiety. A structural model of the MiaE-tRNA complex was simulated based on the crystallographic structures, the biochemical and spectroscopic results. This model reveals that a non-canonical flexible loop of the MiaE enzyme, is likely involved in tRNA recognition and docking processes.
Together, these results pave the way for understanding the mechanisms involved in enzyme-substrate recognition in tRNA modification reactions.


A - Typical modifications of tRNAs (in green), including those at position 37 on the anticodon loop.
B - Details of the A37 hyper-modifications of tRNA catalyzed successively by the MiaA, MiaB and MiaE enzymes.
C - Structure of the MiaE enzyme, the oxygen channel is in red, the cavity housing the tRNA is in light blue. The tRNA is in blue and the modified base is in green.