Crystal structure of archaeosine tRNA-guanine transglycosylase

Archaeosine tRNA-guanine transglycosylase (ArcTGT) catalyzes the exchange of guanine at position 15 in the D-loop of archaeal tRNAs with a free 7-cyano-7-deazaguanine (preQ(0)) base, as the first step in the biosynthesis of an archaea-specific modified base, archaeosine (7-formamidino-7-deazaguanosine). We determined the crystal structures of ArcTGT from Pyrococcus horikoshii at 2.2 A resolution and its complexes with guanine and preQ(0), at 2.3 and 2.5 A resolutions, respectively. The N-terminal catalytic domain folds into an (alpha/beta)(8) barrel with a characteristic zinc-binding site, showing structural similarity with that of the bacterial queuosine TGT (QueTGT), which is involved in queuosine (7-[[(4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]methyl]-7-deazaguanosine) biosynthesis and targets the tRNA anticodon. ArcTGT forms a dimer, involving the zinc-binding site and the ArcTGT-specific C-terminal domain. The C-terminal domains have novel folds, including an OB fold-like "PUA domain", whose sequence is widely conserved in eukaryotic and archaeal RNA modification enzymes. Therefore, the C-terminal domains may be involved in tRNA recognition. In the free-form structure of ArcTGT, an alpha-helix located at the rim of the (alpha/beta)(8) barrel structure is completely disordered, while it is ordered in the guanine-bound and preQ(0)-bound forms. Structural comparison of the ArcTGT.preQ(0), ArcTGT.guanine, and QueTGT.preQ(1) complexes provides novel insights into the substrate recognition mechanisms of ArcTGT.     

The Escherichia coli tRNA-guanine transglycosylase can recognize and modify DNA.

tRNA-guanine transglycosylase (TGT) catalyzes the exchange of queuine (or a precursor) for guanine 34 in tRNA. The minimal RNA recognition motif for TGT has been found to involve a UGU sequence in the anticodon loop of the queuine-cognate tRNAs. Recent studies have shown that the enzyme is capable of recognizing the UGU sequence in alternative contexts (Kung, F. L., Nonekowski, S., and Garcia, G. A. (2000) RNA 6, 233-244) and have investigated the role of the first U of the UGU sequence in tRNA recognition by TGT (Nonekowski, S. T., and Garcia, G. A. (2001) RNA 7, 1432-1441). The TGT reaction involves the breakage and re-formation of a glycosidic bond. To rule out a potential chemical mechanism involving the 2'-hydroxyl at position 34, we synthesized and evaluated an RNA minihelix with 2'-deoxy-G at 34. The high level of activity exhibited by this analogue indicates that the 2'-hydroxyl of G(34) is not required for catalysis. Furthermore, we find that TGT can recognize analogues composed entirely of DNA, but only when 2'-deoxyuridines replace the thymidines in the DNA. The requirement for uridine bases for recognition is perhaps not surprising given the UGU recognition motif for TGT. However, it is not clear if the uracil requirement is due to specific recognition by TGT or due to the effect of uracils on the conformation of the oligonucleotide.     

Absence of tRNA-guanine transglycosylase in a human colon adenocarcinoma cell line.

Queuosine (Q), found exclusively in the first position of the anticodons of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr), is synthesized in eucaryotes by a base-for-base exchange of queuine, the base of Q, for guanine at tRNA position 34. This reaction is catalyzed by the enzyme tRNA-guanine transglycosylase (EC 2.4.2.29). We measured the specific release of queuine from Q-5'-phosphate (queuine salvage) and the extent of tRNA Q modification in 6 human tumors carried as xenografts in immune-deprived mice. Q-deficient tRNA was found in 3 of the tumors but it did not correlate with diminished queuine salvage. The low tRNA Q content of one tumor, the HxGC3 colon adenocarcinoma, prompted us to examine a HxGC3-derived cell line, GC3/M. GC3/M completely lacks Q in its tRNA and measurable tRNA-guanine transglycosylase activity; the first example of a higher eucaryotic cell which lacks this enzyme. Exposure of GC3/M cells to 5-azacytidine induces the transient appearance of Q-positive tRNA. This result suggests that at least one allele of the transglycosylase gene in GC3/M cells may have been inactivated by DNA methylation. In clinical samples, we found Q-deficient tRNA in 10 of 46 solid tumors, including 2 of 13 colonic carcinomas.     

tRNA recognition by tRNA-guanine transglycosylase from Escherichia coli: the role of U33 in U-G-U sequence recognition.

In eubacteria, the biosynthesis of queuine, a modified base found in the wobble position (#34) of tRNAs coding for Tyr, His, Asp, and Asn, occurs via a multistep pathway. One of the key enzymes in this pathway, tRNA-guanine transglycosylase (TGT), exchanges the genetically encoded guanine at position 34 with a queuine precursor, preQ1. Previous studies have identified a minimal positive RNA recognition motif for Escherichia coli TGT consisting of a stable minihelix that contains a U-G-U sequence starting at the second position of its seven base anticodon loop. Recently, we reported that TGT was capable of recognizing the U-G-U sequence outside of this limited structural context. To further characterize the ability of TGT to recognize the U-G-U sequence in alternate contexts, we constructed mutants of the previously characterized E. coli tRNA(Tyr) minihelix. The U-G-U sequence was shifted to various positions within the anticodon loop of these mutants. Characterization of these analogs demonstrates that in addition to the normal U33G34U35 position, TGT can also recognize the U34G35U36 analog (UGU(+1)). The other analogs were not active. This indicates that the recognition of the U-G-U sequence is not strictly dependent upon its position relative to the stem. In E. coli, the full-length tRNA with a U34G35U36 anticodon sequence is one of the isoacceptors that codes for threonine. We found that TGT is able to recognize tRNA(Thr(UGU)) but only in the absence of a uridine at position 33. U33, an invariant base present in all tRNAs, has been shown to strongly influence the conformation of the anticodon loop of certain tRNAs. We find that mutation of this base confers on TGT the ability to recognize U34G35U36, and suggests that loop conformation affects recognition. The fact that the other analogs were not active indicates that although TGT is capable of recognizing the U-G-U sequence in additional contexts, this recognition is not indiscriminate.     

Glutamate versus glutamine exchange swaps substrate selectivity in tRNA-guanine transglycosylase: insight into the regulation of substrate selectivity by kinetic and crystallographic studies

Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of guanine in the wobble position of particular tRNAs by the modified base preQ₁. In vitro, however, the enzyme is also able to insert the immediate biosynthetic precursor, preQ₀, into those tRNAs. This substrate promiscuity is based on a peptide switch in the active site, gated by the general acid/base Glu235. The switch alters the properties of the binding pocket to allow either the accommodation of guanine or preQ₁. The peptide conformer recognising guanine, however, is also able to bind preQ₀. To investigate selectivity regulation, kinetic data for Zymomonas mobilis Tgt were recorded. They show that selectivity in favour of the actual substrate preQ₁ over preQ₀ is not achieved by a difference in affinity but via a higher turnover rate. Moreover, a Tgt(Glu235Gln) variant was constructed. The mutation was intended to stabilise the peptide switch in the conformation favouring guanine and preQ₀ binding. Kinetic characterisation of the mutated enzyme revealed that the Glu235Gln exchange has, with respect to all substrate bases, no significant influence on k_cat. In contrast, K_M(preQ₁) is drastically increased, while K_M(preQ₀) seems to be decreased. Hence, regarding k_cat/K_M as an indicator for catalytic efficiency, selectivity of Tgt in favour of preQ₁ is abolished or even inverted in favour of preQ₀ for Tgt(Glu235Gln). Crystal structures of the mutated enzyme confirm that the mutation strongly favours the binding pocket conformation required for the accommodation of guanine and preQ₀. The way this is achieved, however, significantly differs from that predicted based on crystal structures of wild-type Tgt.     

Evolution of eukaryal tRNA-guanine transglycosylase: insight gained from the heterocyclic substrate recognition by the wild-type and mutant human and Escherichia coli tRNA-guanine transglycosylases

The enzyme tRNA-guanine transglycosylase (TGT) is involved in the queuosine modification of tRNAs in eukarya and eubacteria and in the archaeosine modification of tRNAs in archaea. However, the different classes of TGTs utilize different heterocyclic substrates (and tRNA in the case of archaea). Based on the X-ray structural analyses, an earlier study [Stengl et al. (2005) Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. Chembiochem, 6, 1926-1939] has made a compelling case for the divergent evolution of the eubacterial and archaeal TGTs. The X-ray structure of the eukaryal class of TGTs is not known. We performed sequence homology and phylogenetic analyses, and carried out enzyme kinetics studies with the wild-type and mutant TGTs from Escherichia coli and human using various heterocyclic substrates that we synthesized. Observations with the Cys145Val (E. coli) and the corresponding Val161Cys (human) TGTs are consistent with the idea that the Cys145 evolved in eubacterial TGTs to recognize preQ(1) but not queuine, whereas the eukaryal equivalent, Val161, evolved for increased recognition of queuine and a concomitantly decreased recognition of preQ(1). Both the phylogenetic and kinetic analyses support the conclusion that all TGTs have divergently evolved to specifically recognize their cognate heterocyclic substrates.     

Characterization of cDNA encoding the human tRNA-guanine transglycosylase (TGT) catalytic subunit

Queuosine (Q) is a 7-deazaguanosine found in the first position of the anticodon of tRNAs that recognize NAU and NAC codons (Tyr, Asn, Asp and His). Eukaryotes synthesize Q by the base-for-base exchange of queuine (Q base) for guanine in the unmodified tRNA, a reaction catalyzed by TGT. A search of the human EST database for sequences with significant homology to the well studied TGT from Escherichia coli identified several candidates for full-length (1.3-1.4 kb) cDNA clones. Three candidate cDNA clones, available from IMAGE Consortium, LLNL, (Lennon et al., 1996, Genomics 33, 151-152) were obtained: IMAGE Clone Id Nos. 611146, 1422928, and 72154. Here we report the complete sequences of these clones. IMAGE:72154 contains an ORF encoding a 44 kDa polypeptide with high homology to bacterial TGTs and was subcloned into the mammalian expression vector pMAMneo-Cat. When this construct was transfected into the TGT-negative cell line, GC(3)/c1 (Gündüz et al., 1992, Biochim. Biophys. Acta 1139, 229-238), it restored the ability of the cells to form Q-containing tRNA. This TGT cDNA sequence is encoded in human chromosome 19 clone CTC-539A10 (GenBank accession no. AC011475), enabling determination of the exon-intron boundaries for the TGT gene. The sequence of IMAGE:611146 is 5'-truncated by 76 bp compared to that from IMAGE:72154 and, except for two differences in the 3'-non-coding region, the remainder of the sequence is identical to that of IMAGE:72154. IMAGE:1422928 is a 1390 bp chimera: the 5'-portion, bp 1-708, is identical to a genomic DNA sequence from chromosome 15 (GenBank accession no. AC067805, bp 148976-149683); the 3'-end, bp 726-1390, is identical to the 3'-end of the TGT cDNA sequence from IMAGE:611146.     

Crystal structure of a brominated RNA helix with four mismatched base pairs: An investigation into RNA conformational variability.

The X-ray crystal structure of a brominated RNA helix with four mismatched base pairs and sequence r(UG(Br)C(Br)CAGUUCGCUGGC)(2) was determined to 2.1 A using the methods of multiwavelength anomalous diffraction (MAD) applied to the bromine K-absorption edge. There are three molecules in the asymmetric unit with unique crystal-packing environments, revealing true conformational variability at high resolution for this sequence. The structure shows that the sequence itself does not define a consistent pattern of solvent molecules, with the exception of the mismatched base pairs, implying that specific RNA-protein interactions would occur only with the nucleotides. There are a number of significant tertiary interactions, some of which are a result of the brominated base pairs and others that are directly mediated by the RNA 2' hydroxyl groups. The mismatched base pairs exhibit a solvent network as well as a stacking pattern with their nearest neighbors that validate previous thermodynamic analysis.     

A new target for shigellosis: rational design and crystallographic studies of inhibitors of tRNA-guanine transglycosylase

Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hyper-modification of cognate tRNAs leading to the exchange of G34 at the wobble position in the anticodon loop by preQ1 (2-amino-5-(aminomethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one) as part of the biosynthesis of queuine (Q). Mutation of the tgt gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium, revealing TGT as a new target for the design of potent drugs against Shigellosis. The X-ray structure of Zymomonas mobilis TGT in complex with preQ1 was used to search for new putative inhibitors with the computer program LUDI. An initial screen of the Available Chemical Directory, a database compiled from commercially available compounds, suggested several hits. Of these, 4-aminophthalhydrazide (APH) showed an inhibition constant in the low micromolar range. The 1.95 A crystal structure of APH in complex with Z. mobilis TGT served as a starting point for further modification of this initial lead.     

The RNA-binding PUA domain of archaeal tRNA-guanine transglycosylase is not required for archaeosine formation

Bacterial tRNA-guanine transglycosylase (TGT) replaces the G in position 34 of tRNA with preQ(1), the precursor to the modified nucleoside queuosine. Archaeal TGT, in contrast, substitutes preQ(0) for the G in position 15 of tRNA as the first step in archaeosine formation. The archaeal enzyme is about 60% larger than the bacterial protein; a carboxyl-terminal extension of 230 amino acids contains the PUA domain known to contact the four 3'-terminal nucleotides of tRNA. Here we show that the C-terminal extension of the enzyme is not required for the selection of G15 as the site of base exchange; truncated forms of Pyrococcus furiosus TGT retain their specificity for guanine exchange at position 15. Deletion of the PUA domain causes a 4-fold drop in the observed k(cat) (2.8 x 10(-3) s(-1)) and results in a 75-fold increased K(m) for tRNA(Asp)(1.2 x 10(-5) m) compared with full-length TGT. Mutations in tRNA(Asp) altering or abolishing interactions with the PUA domain can compete with wild-type tRNA(Asp) for binding to full-length and truncated TGT enzymes. Whereas the C-terminal domains do not appear to play a role in selection of the modification site, their relevance for enzyme function and their role in vivo remains to be discovered.     

Purification,crystallization, and preliminary X-ray diffraction studies of tRNA-guanine transglycosylase from Zymomonas mobilis

The tRNA modifying enzyme tRNA–guanine transglycosylase (Tgt) catalyzes the exchange of guanine in the first position of the anticodon with the queuine precursor 7-aminomethyl-7-deazaguanine. Tgt from Zymomonas mobilis has been purified by crystallization and further recrystallized to obtain single crystals suitable for x-ray diffraction studies. Crystals were grown by vapor diffusion/gel crystallization methods using PEG 8,000 as precipitant. Macroseeding techniques were employed to produce large single crystals. The crystals of Tgt belong to the monoclinic space group C2 with cell constants a = 92.1 Å, b = 65.1 Å, c = 71.9 Å, and β = 97.5°, and contain one molecule per asymmetric unit. A complete diffraction data set from one native crystal has been obtained at 1.85 Å resolution.     

Crystallographic study of inhibitors of tRNA-guanine transglycosylase suggests a new structure-based pharmacophore for virtual screening

The enzyme tRNA-guanine transglycosylase (TGT) is involved in the pathogenicity of Shigellae. As the crystal structure of this protein is known, it is a putative target for the structure-based design of inhibitors. Here we report a crystallographic study of several new ligands exhibiting a 2,6-diamino-3H-quinazolin-4-one scaffold, which has been shown recently to be a promising template for TGT-inhibitors. Crystal structure analysis of these complexes has revealed an unexpected movement of the side-chain of Asp102. A detailed analysis of the water network disrupted by this rotation has lead to the derivation of a new composite pharmacophore. A virtual screening has been performed based on this pharmacophore hypothesis and several new inhibitors of micromolar binding affinity with new skeletons have been discovered.