Homodimer Architecture of QTRT2, the Noncatalytic Subunit of the Eukaryotic tRNA-Guanine Transglycosylase

Abstract

The bacterial enzyme tRNA-guanine transglycosylase (TGT) is involved in the biosynthesis of queuosine, a modified nucleoside present in the anticodon wobble position of tRNA, tRNA, tRNA, and tRNA. Although it forms a stable homodimer endowed with two active sites, it is, for steric reasons, able to bind and convert only one tRNA molecule at a time. In contrast, its mammalian counterpart constitutes a heterodimer consisting of a catalytic and a noncatalytic subunit, termed QTRT1 and QTRT2, respectively. Both subunits are homologous to the bacterial enzyme, yet only QTRT1 possesses all the residues required for substrate binding and catalysis. In mice, genetic inactivation of the TGT results in the uncontrolled oxidation of tetrahydrobiopterin and, accordingly, phenylketonuria-like symptoms. For this reason and because of the recent finding that mammalian TGT may be utilized for the treatment of multiple sclerosis, this enzyme is of potential medical relevance, rendering detailed knowledge of its biochemistry and structural architecture highly desirable. In this study, we performed the kinetic characterization of the murine enzyme, investigated potential quaternary structures of QTRT1 and QTRT2 via noncovalent mass spectrometry, and, finally, determined the crystal structure of the murine noncatalytic TGT subunit, QTRT2. In the crystal, QTRT2 is clearly present as a homodimer that is strikingly similar to that formed by bacterial TGT. In particular, a cluster of four aromatic residues within the interface of the bacterial TGT, which constitutes a "hot spot" for dimer stability, is present in a similar constellation in QTRT2.

Are Metabolites From the Gut Microbiota Capable of Regulating Epigenetic Mechanisms in the Human Parasite Entamoeba histolytica?

“…Both h QTRT1 and h QTRTD1 co-localize in mitochondria ( Boland et al, 2009 ). The crystal structure of h QTRTD1 (also called QTRT2) revealed that h QTRTD1 forms a homodimer with striking similarity to that of bacterial TGT ( Behrens et al, 2018 ). Recently, the crystal structure of hTGT in its heterodimeric form and in complex with a 25-mer stem loop RNA has been established ( Sievers et al, 2021 ).…”

Crystal Structure of the Human tRNA Guanine Transglycosylase Catalytic Subunit QTRT1

“…This quaternary structure has been confirmed to be essential for catalyzing the incorporation of queuine into tRNA [ 31 ]. Recently, the crystal structure of the noncatalytic mouse QTRT2 subunit has been solved and found to confirm the prior suggestion that this subunit is incapable to catalyze the guanine to queuine exchange reaction as catalytic residues, and the residues involved in substrate coordination are largely replaced by chemically different amino acids and thus are extremely unlikely to facilitate queuine binding or even the transfer reaction [ 32 ]. In contrast, alignment of the human QTRT1 sequence with the Z. mobilis TGT reveals this subunit to be likely catalytically active with relevant residues in place.…”

Structural and Biochemical Investigation of the Heterodimeric Murine tRNA-Guanine Transglycosylase

“…■ METHODS Gene Expression and Mutagenesis, Protein Production, and Purification. The recombinant, separately performed overexpression of murine QTRT1 and QTRT2 genes as well as the purification of the respective gene products were carried out, as described by Behrens et al 19 The site-directed mutagenesis of the QTRT2 gene was carried out via the QuikChange Lightning kit (Agilent) according to the vendor's instructions. For DNA manipulation, plasmids were prepared using the peqGOLD plasmid miniprep kit (PEQLAB).…”

“…30,33,35 There is, however, no evidence about the importance of QTRT2 residue Tyr354 for dimer stability since a corresponding aromatic residue does not exist in Z. mobilis TGT. The crystal structure of murine QTRT2, published in 2018, 19 reveals that this residue not only seems to be of relevance for the stability of the TGT heterodimer but also for the stability of the QTRT2 homodimer. Here, it forms a subunit-bridging H bond to the main chain amide of Gln48′.…”

“…Accordingly, instead of the preQ 1 base, the eukaryotic TGT (EC 2.4.2.64) has to accept queuine as a substrate in order to insert it into position 34 of tRNA Asp , tRNA Asn , tRNA Tyr , and tRNA His . In contrast to its bacterial counterpart, the eukaryotic TGT is a heterodimer with both of its subunits, QTRT1 and QTRT2, closely resembling the bacterial TGT monomer. − However, only QTRT1 is endowed with an active center, whereas the non-catalytic QTRT2 obviously supports the potent binding and the correct orientation of the tRNA substrate.…”