Identification of the Rate-Determining Step of tRNA-Guanine Transglycosylase from Escherichia coli

Abstract

The modified RNA base queuine (7-(4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanine) occurs in tRNA via a unique base exchange process catalyzed by tRNA-guanine transglycosylase (TGT). Previous studies have suggested the intermediacy of a covalent TGT-RNA complex. To exist on the reaction pathway, this covalent complex must be both chemically and kinetically competent. Chemical competence has been demonstrated by the crystal structure studies of Xie et al. (Nature Structural Biology (2003) 10, 781-788); however, evidence of kinetic competence had not yet been established. The studies reported here unequivocally demonstrate that the TGT-RNA covalent complex is kinetically capable of occurring on the TGT reaction pathway. These studies further suggest that product RNA dissociation from the enzyme is overall rate-limiting in the steady-state. Interestingly, studies comparing RNA with a 2′-deoxyriboside at the site of modification suggest a role for the 2′-hydroxyl group in stabilizing the growing negative charge on the nucleophilic aspartate (264) as the glycosidic bond to the aspartate is broken during covalent complex breakdown.Over one hundred chemically distinct modified bases are known to occur in RNA, the majority of which occur in tRNA (1). Of these, queuine (Q) stands out for several reasons. Structurally, queuine is the only modified base that is not a purine or pyrimidine analog. Instead, queuine features a pyrrolo-pyrimidine heterocyclic scaffold that is further elaborated through exocyclic chemical modifications. Perhaps most interestingly, the mechanism of incorporation of queuine into tRNA is unique. The queuine base is post-transcriptionally introduced into tRNA via a transglycosylation reaction that is catalyzed by tRNA-guanine transglycosylase (TGT) (Figure 1). Among the known modified bases, only pseudouridine is installed in an analogous manner whereby pseudouridine synthase breaks and reforms the glycosidic bond to the uracil (2).As with pseudouridine synthase, the chemical mechanism of the TGT reaction has been studied for some time. Two distinct mechanisms have been proposed and subsequently investigated for the TGT catalyzed base-exchange reaction. It was first envisaged that a dissociative mechanism, involving the intermediacy of an oxocarbonium ion, could drive the cleavage of † This work was supported in part by the National Institutes of Health (GM065489, GAG; GM07767, JDK trainee), and the University the glycosidic bond leading to the incorporation of the modified base. Alternatively, and equally plausible, the TGT reaction may proceed through a covalent TGT-RNA intermediate (3) in an associative mechanism similar to that of retaining glycosyl hydrolases (4) ( Figure 2A). In fact, biochemical studies from our laboratory have implicated the associative mechanism and the involvement of two active site aspartate residues in the reaction (5,6). Consistent with the associative mechanism, we have previously shown that the E. coli TGT follows ping-pong kinetics (7), with...

Differential heterocyclic substrate recognition by, and pteridine inhibition of E. coli and human tRNA-guanine transglycosylases

“…However the K M values are much closer (within an order of magnitude), with the exception of the quadruple mutant, which is 100-fold higher than wild-type. Previously, we have shown that some mutants of C145 exhibit increased k cat and K M values [15], consistent with our determination that product release is rate-limiting [20], i.e., poorer binding due to a faster off-rate would result in faster k cat and larger K M . Additional mutations of the other residues cause decreases in k cat and increases in K M to relatively smaller extents (Table 2).…”

Characterization of the human tRNA-guanine transglycosylase: Confirmation of the heterodimeric subunit structure

“…Derived from fit to the following equation: To determine which subunit (hQTRT1 or hQTRTD1) is actually responsible for the transglycosylase activity, two human TGT mutants, ht-hQTRT1(D279N) d hQTRTD1 and ht-hQTRT1 d hQTRTD1(E272Q), were expressed and purified. The corresponding residue, aspartate 264, in the E. coli TGT is the nucleophilic catalyst that forms the covalent RNA-TGT intermediate (Kittendorf et al 2003;Xie et al 2003;Garcia et al 2009). Mutation to anything other than glutamate yields catalytically inactive protein that is structurally unaltered (i.e., it folds correctly and binds, noncovalently to tRNA).…”

Biosynthesis of pyrrolopyrimidines

“…Moreover, the captured covalent intermediate is converted to the product by soaking preQ 1 into the crystals, demonstrating its intermediacy in the catalytic cycle [73]. Site-directed and kinetic studies have confirmed the role of the Asp and have demonstrated kinetic competence for the covalent intermediate [74,75]. …”