Mechanistic investigations of the pseudouridine synthase RluA using RNA containing 5-fluorouridine

Abstract

The pseuoduridine synthases (psi synthases) isomerize uridine (U) to pseudouridine (psi) in RNA, and they fall into five families that share very limited sequence similarity but have the same overall fold and active-site architecture, including an essential Asp. The mechanism by which the psi synthases operate remains unknown, and mechanistic work has largely made use of RNA containing 5-fluorouridine (f5U) in place of U. The psi synthase TruA forms a covalent adduct with such RNA, and heat disruption of the adduct generates a hydrated product of f5U, which was reasonably concluded to result from the hydrolysis of an ester linkage between the essential Asp and f5U. In contrast, the psi synthase TruB, which is a member of a different family, does not form an adduct with f5U in RNA but catalyzes the rearrangement and hydration of the f5U, which labeling studies with [18O]water showed does not result from ester hydrolysis. To extend the line of mechanistic investigation to another family of psi synthases and an enzyme that makes an adduct with f5U in RNA, the behavior of RluA toward RNA containing f5U was examined. Stem-loop RNAs are shown to be good substrates for RluA. Heat denaturation of the adduct between RluA and RNA containing f5U produces a hydrated nucleoside product, and labeling studies show that hydration does not occur by ester hydrolysis. These results are interpreted in light of a consistent mechanistic scheme for the handling of f5U by psi synthases.

Figure 1

Proposed mechanisms for the Ψ synthase reaction. A, the “Michael mechanism” in which the essential Asp nucleophilically adds to the pyrimidine ring. B, the “acylal” mechanism, in which the essential Asp nucleophilically adds to the ribose ring either by a concerted (upper path) or stepwise (lower path) process. The stereochemistry of the rearranged uridine is based on that observed in the cocrystal of TruB and [f⁵U]TSL (11).

Figure 2

Possible reactions between Ψ synthases and f⁵U in RNA showing the predicted labeling in [¹⁸O]water (denoted by the half-filled O). A, the initial Michael adduct between f⁵U and the catalytic Asp undergoes ester hydrolysis. B, the ultimate Michael adduct between f⁵U and the catalytic Asp undergoes ester hydrolysis. C, the catalytic Asp acts as a general base in the direct hydration of rearranged f⁵U. D, the catalytic Asp adds to the rearranged f⁵U to make a covalent adduct, which can then suffer ester hydrolysis (downwards on the right) or elimination (reversal of the top reaction) followed by release into solution and spontaneous hydration (downwards on the left). Ester hydrolysis results in the labeling of the catalytic Asp (A, B, and right side of D), while direct hydration (C and left side of D) labels the nucleoside.

Figure 3

Stem-loop structures in natural substrates for E. coli RluA that contain the isomerized uridine residue (underlined), U32 in the anticodon stem-loops (ASLs) of four tRNAs and U746 in a 23S rRNA stem-loop (RSL). The ASLs span residues 27–43 of tRNA, and RSL spans residues 737–760 of 23S rRNA.

Figure 4

HPLC analysis of ASL before (solid) and after (dashed) incubation with RluA. A, partial HPLC trace of intact ASL. The major peaks are substrate ASL (with U) at 15 min and product ASL (with Ψ) at 14 min. A small amount of substrate ASL remains in the product trace; the small peak at 11 minutes is a component of the reaction buffer. B, partial HPLC trace of the nucleosides from the complete digestion of ASL. The substrate trace shows U (5.00 min) at the expected ratio to C (3.05 min); the product trace shows diminished U and the appearance of Ψ (2.08 min) at the expected ratio relative to the other nucleosides. The G and A peaks (not shown) are not changed in intensity upon incubation with RluA, as expected.

Figure 5

Briggs-Haldane plot of the kinetic data for RluA with ASL as substrate. The enzyme concentration was 50 nM. Each point is the average of at least two independent determinations; error bars are omitted when the error is smaller than the size of data point.

Figure 6

SDS-PAGE analysis of the formation of the adduct between RluA and [f⁵U]ASL. A, the gel shift of wild-type RluA upon incubation with [f⁵U]ASL indicates adduct formation (lanes d and e). Heating disrupts the adduct (lanes b and c), which does not form with D64A RluA (lane h). B, The time course for the formation of the adduct between wild-type RluA and [f⁵U]ASL. Adduct formation was monitored in a reaction containing a five fold excess of [f⁵U]ASL over RluA. The adduct is detected at 0.5 min, and the reaction is half complete by 4 min.

Figure 7

HPLC analysis of [f⁵U]ASL before (solid) and after (dashed) incubation with RluA and heat denaturation of the adduct. A, intact stem-loop showing an increased retention time for product [f⁵U]ASL. B, after digestion to free nucleosides; f⁵U elutes at 6.67 min, and the modified f⁵U product elutes at 2.64 min.

Figure 8

Labeling studies to probe for ester hydrolysis during the decomposition of the adduct between RluA and [f⁵U]ASL. The adduct was formed with a 2-fold excess of RluA and then heat-disrupted, and the protein and RNA were separately digested and subjected to MALDI-MS analysis. A, Partial mass spectra of the tryptic digest of RluA showing the tryptic peptide ⁵³Leu-Lys⁷⁷ (2700.4 m/z predicted) containing Asp-64 (left) and the predicted mass distribution for ester hydrolysis of a Michael adduct (right); top, RluA incubated in unlabeled buffer with [f⁵U]ASL; middle, RluA incubated with [f⁵U]ASL in buffer containing [¹⁸O]water (50%), showing no ¹⁸O incorporation into the peptide, which does not match the prediction for ester hydrolysis; bottom, RluA incubated in unlabeled buffer with [f⁵U]ASL with trypsinolysis in buffer containing [¹⁸O]water (50%). B, Partial mass spectra of [f⁵U]ASL after incubation with RluA and digestion with RNase T₁; top, incubation in unlabeled buffer; bottom, incubation in buffer containing [¹⁸O]water (50%), showing ¹⁸O incorporation into the hydrated [f⁵U]ASL (50% of the product contains ¹⁶O and 50% contains ¹⁸O and is therefore shifted +2 m/z). The small [M]⁺ peak observed could be due to amination rather than hydration since ammonium chloride (100 mM) was present in the reaction mixture; however, the [M]⁺ was still present when ammonium chloride was replaced with sodium chloride (data not shown), so it more likely arises from the high laser power necessary to obtain the mass spectra of the oligonucleotides.