The enzyme tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) catalyzes a posttranscriptional transglycosylation reaction involved in the incorporation of the modified base queuine [Q, 7-(4,5-cis-dihydroxy-2-cyclopenten-1-ylaminomethyl)-7-deazaguanine] into tRNA. Previously, the crystal structure of the TGT from Zymomonas mobilis was solved in complex with preQ(1) (the substrate for the eubacterial TGT) [Romier et al. (1996) EMBO J. 15, 2850-2857]. An aspartate residue at position 102 (position 89 in the Escherichia coli TGT) was proposed to play a nucleophilic role in an associative catalytic mechanism. Although this is an attractive and precedented mechanism, a dissociative mechanism is equally plausible. In a dissociative mechanism, aspartate 89 would provide electrostatic stabilization of an oxocarbenium ion intermediate that is formed by dissociation of guanine. To clarify the nature of the catalytic mechanism of TGT, we have generated and characterized four mutations of aspartate 89 in the E. coli TGT (alanine, asparagine, cysteine, and glutamate). All four mutant TGTs were able to noncovalently bind tRNA, but only the glutamate mutant was able to form a stable complex with the RNA substrate under denaturing conditions that was comparable to wild type. Furthermore, the glutamate mutant was the only mutant TGT that demonstrated significant activity. Kinetic parameters were determined for this enzyme and shown to be comparable to wild type, revealing that the enzyme is considerably tolerant of the positioning of the carboxylate. Under conditions of high enzyme concentrations and long time courses, the alanine, asparagine, and cysteine mutants showed very low levels (ca. 10(3)-fold lower than wild type) of activity that were linear with respect to enzyme concentration and dependent upon pH in a fashion similar to that of the wild type. However, the observed initial velocities were too low to accurately determine k(cat) and K(m) values. We hypothesize that the activity observed for these mutants is most likely derived from host strain TGT (wt) contamination. These results are most consistent with aspartate 89 acting as a nucleophile in an associative catalytic mechanism.

tRNAs contain a large number of modified nucleosides (1). One of the more elaborately modified nucleosides is queuine (7-(4,5-cis-dihydroxy-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanine). tRNA-guanine transglycosylase (TGT) 1 catalyzes the exchange of queuine (or a precursor) for guanine 34 in the anticodon of certain tRNAs. The minimal RNA recognition motif for TGT has been found to involve a UGU sequence in the anticodon loop of the queuine-cognate tRNAs (tyrosine, aspartate, asparagine, and histidine) (2, 3). The UGU sequence is undeniably the major determinant for tRNA recognition by TGT. However, provided that TGT can position the UGU sequence in the active site in the proper orientation, the UGU sequence need not reside in the anticodon loop to be recognized. Recent studies have shown that TGT can recognize the UGU sequence in at least 2 additional minihelical contexts, at the base of the T⌿C stem in yeast tRNA Phe and in the anticodon position in the absence of U33 (4, 5). Thus, tRNA recognition by TGT is more flexible than previously believed. This observation prompted an examination of the ability of TGT to recognize RNAs containing modifications of the UGU sequence. The initial studies that identified the UGU sequence as the major identity element utilized only canonical base (C, G, A, U) replacements (2, 3). Previous experiments have demonstrated that a DNA analogue of an RNA minihelix corresponding to the anticodon arm of Escherichia coli tRNA Tyr , ECYMH (dEC-YMH) was inactive.2 However, there are two fundamental differences between RNA and DNA, the lack of the 2Ј-hydroxyls and the presence of thymidine rather than uracil in DNA. Therefore, dECYMH has a TGT sequence rather than a UGU sequence.Given the importance of the UGU sequence in TGT recognition, it is possible that the inactivity of dECYMH was due to the presence of thymidine rather than the loss of the 2Ј-hydroxyls. To investigate the role of the 2Ј-hydroxyl in TGT recognition and catalysis, we have studied a deoxyguanosine 34 analogue of ECYMH (Fig. 1, dG 34 ECYMH). This analogue is a substrate for TGT with less than a 10-fold reduction in activity. To further probe the ability of TGT to recognize RNA analogues lacking the 2Ј-hydroxyl, modified DNA analogues of the previously described minihelix ECYMH (2, 6) ( Fig. 1, dUdECYMH) and the alternative TGT minihelical substrates UGU ϩ1 (dUdUGU ϩ1 ) (4) and SCFMH(T⌿C) (dUdSCFMH(T⌿C)) (5) were synthesized and characterized. These analogues (containing deoxyuracil (dU) bases rather than thymidine bases) all serve as substrates for TGT, indicating that the tRNA-guanine transglycosylase from E. coli can recognize and modify DNA.

tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the posttranscriptional modification of tRNA across the three kingdoms of life. In eukaryotes and eubacteria, TGT is involved in the introduction of queuine into the anticodon of the cognate tRNAs. In archaebacteria, TGT is responsible for the introduction of archaeosine into the D-loop of the appropriate tRNAs. The tRNA recognition patterns for the eubacterial (Escherichia coli) TGT have been studied. These studies are all consistent with a restricted recognition motif involving a U-G-U sequence in a seven-base loop at the end of a helix. While attempting to investigate the potential of negative recognition elements in noncognate tRNAs via the use of chimeric tRNAs, we have discovered a second recognition site for the E. coli TGT in the TpsiC arm of in vitro-transcribed yeast tRNA(Phe). Kinetic analyses of synthetic mutant oligoribonucleotides corresponding to the TpsiC arm of the yeast tRNA(Phe) indicate that the specific site of TGT action is G53 (within a U-G-U sequence at the transition of the TpsiC stem into the loop). Posttranscriptional base modifications in tRNA(Phe) block recognition by TGT, most likely due to a stabilization of the tRNA structure such that G53 is inaccessible to TGT. These results demonstrate that TGT can recognize the U-G-U sequence within a structural context that is different than the canonical U-G-U in the anticodon loop of tRNA(Asp). Although it is unclear if this second recognition site is physiologically relevant, this does suggest that other RNA species could serve as substrates for TGT in vivo.

tRNA guanine transglycosylase (TGT) is a tRNA-modifying enzyme which catalyzes the posttranscriptional exchange of guanine in position 34 of tRNA(Y,H,N,D) with the modified base queuine in eukaryotes or its precursor, preQ(1) base, in eubacteria. Thus, TGT must recognize the guanine in tRNA and the free base queuine or preQ(1) to catalyze this exchange. The crystal structure of Zymomonas mobilis TGT with preQ(1) bound suggests that a key aspartate is critically involved in substrate recognition. To explore this, a series of site-directed mutants of D143 in Escherichia coli TGT were made and characterized to investigate heterocyclic substrate recognition. Our data confirm that D143 has significant impact on K(M) of guanine; however, the trend in the K(M) data (D143A < D143N < D143S < D143T) is unexpected. Computational studies were used to further elucidate the interactions between guanine and the D143 mutants. A homology model of E. coli TGT was created, and the role of D143 was investigated by molecular dynamic simulations of guanine bound to the wild-type and D143-mutant TGTs. To validate the model systems against our kinetic data, free energies of binding were fit using the linear interaction energy (LIE) method. This is a unique application of the LIE method because the same ligand is bound to several mutant proteins rather than one protein binding several ligands. The atomic detail gained from the simulations provided a better understanding of the binding affinities of guanine with the mutant TGTs, revealing that water molecules enter the active site and hydrogen bond to the ligand and compensate for lost protein-ligand interactions. The trend of binding affinity for wild-type > D143A > D143N > D143S > D143T appears to be directly related to the degree of hydrogen bonding available to guanine in the binding site.