Date on Master's Thesis/Doctoral Dissertation
Mueller, Eugene G.
Committee Co-Chair (if applicable)
Maurer, Muriel C.
Rich, Christine V.
Van Lanen, Steven
Pseudouridine synthases (ΨSs) catalyze the isomerization of uridine (U) in RNA to pseudouridine (Ψ), which is the most common post-transcriptional modification in RNAs and is ubiquitous within all three domains of life. ΨSs are classified into six different families based on sequence alignments and have a universally conserved aspartic acid residue (‘conserved Asp’ for simplicity) that is absolutely essential for activity. RNA containing 5-fluorouridine ([F5U]RNA) has been used as a mechanistic probe. Upon incubation with [F5U]RNA, TruA and RluA get irreversibly inhibited and appear in a protein-RNA adduct band on denaturing PAGE gels, which is consistent with the ‘Michael mechanism’ that proposes a covalent adduct between the conserved Asp and the pyrimidine ring. E. coli TruB does not get inhibited upon incubation with [F5U]RNA, nor does it form an adduct and instead converts F5U into two rearranged, hydrated products. 18O labeling studies with the E. coli ΨSs TruA, TruB, RluA, and TruD show that the hydration of the products of F5U occurs directly from solution and not through the hydrolysis of a Michael adduct. These 18O labeling studies were extended here to Thermotoga maritima TruB (TmTruB) which showed a behavior intermediate between that of E. coli TruB (no observed adduct; multiple turnovers) and RluA and TruA (stoichiometric adduction; single turnover). 18O label is incorporated into RNA directly from solvent during the reaction of [F5U]RNA catalyzed by TmTruB. These results are consistent with the scheme proposed earlier for the handling of F5U by ΨSs. Among examined ΨSs, TmTruB uniquely shows the interesting ability to catalyze the dehydration and rehydration of the products of F5U. The minor product of F5U from the action of ΨSs is an arabino isomer and its generation requires epimerization at C2′, which suggested that the ΨS mechanism may proceed by deprotonation of C2′ to eliminate the uracil and form a glycal intermediate. To test this mechanistic possibility, substrate RNA stem-loops containing [2′-2H]uridine were prepared and used to measure the deuterium kinetic isotope effect on the conversion of U to Ψ. Deuteration at C2′ reduced both Vmax and Vmax/Km for the reaction as catalyzed by TruB (2.54-fold and 3.58-fold, respectively) and RluA (1.79-fold and 2.17-fold, respectively). These results conclusively indicate that the deprotonation of C2′ occurs in a partially rate-determining step in the conversion of U to Ψ, which is consistent with the ‘glycal mechanism’ but inconsistent with the proposed alternatives.
Veerareddygari, Govardhan reddy, "Mechanistic investigations of pseudouridine synthases : a surprising glycal intermediate lies on the reaction pathway." (2014). Electronic Theses and Dissertations. Paper 1734.